Single domain human serum albumin antibodies

Abstract

Provided herein are sdAbs that have binding specificity for human serum albumin comprising a CDR1, CDR2, and a CDR3. Also provided herein are fusion proteins, conjugates, half-life extended cytokines, and inducible cytokine prodrugs comprising the sdAb.

Description

[0001] Attorney Docket No.: 761146.252320

[0002] SINGLE DOMAIN HUMAN SERUM ALBUMIN ANTIBODIES

[0003]

[0001] The present application claims the benefit of U.S. Provisional Application No.63 / 699,262, filed on September 26, 2024, U.S. Provisional Application No.63 / 699,305, filed on September 26, 2024, U.S. Provisional Application No.63 / 699,291, filed on September 26, 2024, and U.S. Provisional Application No.63 / 704,207, filed on October 7, 2024, the entire contents of each of which are incorporated herein by reference.

[0004] 1. BACKGROUND

[0005]

[0001] Inducible cytokine prodrugs, that are conditionally activated in the tumor microenvironment through protease cleavage to release the fully active, native cytokine within the tumor to stimulate a potent anti-tumor immune response, are described in International Publication Nos.: WO2019 / 222294, WO2019 / 222295, WO2019 / 222296, WO2021 / 097376. These prodrugs typically include a native cytokine polypeptide that is attached to a half-life extension element, and typically a cytokine blocking domain, through a protease cleavable linker. For example, IL-2 prodrugs can include a native IL-2 molecule attached through a protease cleavable linker to a half-life extension domain (e.g., anti-human serum albumin antibody binding fragment such as a VH domain) and an IL-2 blocking element (e.g., anti-IL-2 antibody binding fragment, such as a Fab) to block binding of IL-2 to IL-2β / γ receptors on normal tissue in the periphery. Upon cleavage of the protease cleavable linker in the tumor microenvironment, fully active native IL-2 is released within the tumor to stimulate a potent anti-tumor immune response.

[0006]

[0002] Serum albumin (albumin) is one of the most abundant proteins found in plasma of mammals and has a long serum half-life of about 21 days. Waldman et al., (1969), Prog Allergy, 13:1-110. Albumin contributes to maintaining osmotic pressure and also functions as a transporter of range of molecules, such as fatty acids, metabolites, hormones, and toxins. Peters, (1985), Adv. Protein Chem., pp.161-245.

[0007]

[0003] Albumin binds in vivo to the neonatal Fc receptor (FcRn) and this interaction is known to be important for the long plasma half-life of albumin. Chaudhury et al., (2003), The Journal of Experimental Medicine, 197(3):315-322. FcRn is a dual binding receptor, that in addition to albumin, also binds IgG which also has a long serum half-life and protects proteins from intracellular degradation. Id.

[0008]

[0004] Albumin binds to FcRn in a pH-dependent manner, with binding at acidic pH and no binding or release at neutral pH. Ward et al., (2009) Adv. Immunol., 103, 77-115. FcRn is predominantly localized in acidic endosomal compartments and encounters proteins that are taken up by cells (e.g., endothelial cells) by fluid-phase pinocytosis. Proteins taken up by fluid-phase pinocytosis enter early endosomes and then FcRn-positive acidic endosomes. Id. The low pH in the acidic endosomes allows albumin and other FcRn 1

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[0010] ligands to bind to FcRn. Id. Subsequently, FcRn-ligand complexes are recycled back to the cell surface, where exposure to the physiological pH of the blood (~pH 7.4) causes release of the ligands into the circulation. Id. This process is also referred to as FcRn-mediated cellular recycling. Proteins that do not bind to FcRn progress to lysosomes for proteolytic degradation.

[0011]

[0005] The long serum half-life of albumin can be exploited to engineer therapeutic agents with extended serum half-life, typically through chemically or genetically linking the therapeutic agent to albumin or to FcRn-binding variants or fragments of albumin. An alternative approach to engineer therapeutic agents with extended serum half-life, is to link the therapeutic to an albumin binding domain. Such engineered therapeutics can form a complex with albumin and exploit the FcRn-binding of albumin, and the FcRn-mediated cellular recycling pathway, to extend half-life of the therapeutic. As binding of albumin to FcRn is necessary for FcRn-mediated cellular recycling, it is well accepted in the prior art that the albumin binding domains used in this approach must not interfere with the binding of albumin to FcRn. See, e.g., Mester et al., (2021), MAbs, 13(1):1893888. For example, human IgA1 (a protein with a naturally short-half life) was fused to a Streptococcal protein G-derived albumin binding protein to extend the half-life of IgA1 using the human albumin-human FcRn pathway. Id. Further, human albumin is known to consist of three domains, DI, DII and DIII, with DI and DIII being important for FcRn binding. Id. Accordingly, it is well-known from the prior art that albumin binding domains that bind to DII are suitable for indirectly targeting FcRn and that domains that bind DI or DIII should be avoided. Id.

[0012]

[0006] Recent approaches to cancer therapy have included a variety of approaches directed to potentiating the patient's immune response. Cytokines are well-known modulators of immune function and long thought to be suitable as potential cancer therapeutics. But this promise has not been realized largely due to the potent activity of such molecules, undesired toxicity and short half-life of cytokines. The use of extended half-life forms of active cytokines has been avoided as it is well-understood that the short half-life of active cytokines is an important biological mechanism to control systemic activity and toxicity of these potent immunomodulators.

[0013]

[0007] Many potential therapeutic agents, particularly proteins, are limited for clinical use due to inadequate serum half-lives. Such agents could be developed into clinically effective therapeutic agents by utilizing albumin binding domains that bind to albumin. Accordingly, there is a continued need for therapeutics with extended serum half-lives and methods for treating cancer using such prodrugs.

[0014] 2

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[0016] 2. SUMMARY

[0017]

[0008] The disclosure relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof. Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein. Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.

[0018]

[0009] As described and exemplified herein, contrary to the well-accepted knowledge in the art that anti-albumin binding domains that interfere with albumin binding to FcRn are not suitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short half-life therapeutic proteins), the inventors have discovered that sdAbs that compete with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., the in vivo serum half-life) of short half-life therapeutic agents (e.g., proteins, peptides).

[0019]

[0010] The sdAbs disclosed herein comprise a CDR1, CDR2, and a CDR3. The CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions. The CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 5; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. The CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

[0020]

[0011] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 amino acid substitutions.

[0021]

[0012] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions.

[0022]

[0013] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid 3

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[0024] sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0025]

[0014] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0026]

[0015] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0027]

[0016] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0028]

[0017] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0029]

[0018] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0030]

[0019] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a

[0031] 4

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[0033] CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0034]

[0020] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0035]

[0021] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0036]

[0022] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0037]

[0023] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0038]

[0024] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0039]

[0025] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 7; a CDR2 that consists of SEQ ID NO: 38; and a CDR3 that consists of SEQ ID NO: 12.

[0040]

[0026] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 37; a CDR2 that consists of SEQ ID NO: 9; and a CDR3 that consists of SEQ ID NO: 12.

[0041] 5

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[0043]

[0027] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 10; and a CDR3 that consists of SEQ ID NO: 12.

[0044]

[0028] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 11; and a CDR3 that consists of SEQ ID NO: 12.

[0045]

[0029] The sdAb can comprise CDR1 that consists of SEQ ID NO: 1; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 3.

[0046]

[0030] The sdAb can comprise CDR1 that consists of SEQ ID NO: 4; a CDR2 that consists of SEQ ID NO: 5; and a CDR3 that consists of SEQ ID NO: 6.

[0047]

[0031] The sdAbs disclosed herein can compete with neonatal Fc receptor (FcRn) for binding to HSA. The sdAbs can have binding specificity for domain one of human serum albumin.

[0048]

[0032] The sdAb can be a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain. VH and VHH are preferred sdAbs.

[0049]

[0033] Typically, the sdAb is camelid, human, or humanized. Preferably, the sdAb is humanized.

[0050]

[0034] The sdAb’s disclosed herein further comprise one or more framework regions. The framework region can be derived from a human germline.

[0051]

[0035] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 37; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 38; a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and a FR1, a FR2, a FR3, and a FR4. The amino acid residue at position 24 of FR1 can be a serine or an alanine, the amino acid at position 44 of F2 can be a glutamic acid or a glycine, the amino acid at position 45 of F2 can be a arginine or a leucine, and the amino acid at position 79 of F3 is a leucine or a valine. FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

[0052]

[0036] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a FR1, a FR2, a FR3, and a FR4. The amino acid residue at position 24 of FR1 can be a serine or an alanine, the amino acid at position 44 of FR2 can be a aspartic acid or a glycine; the amino acid at position 45 of FR2 can be a arginine or a leucine; the amino acid at position 49 of FR2 can be a serine or alanine, the amino acid at position 78 of FR3 can be a leucine or a phenylalanine, and the amino acid at position 79 of F3 can be a asparagine or a lysine. The FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

[0053] 6

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[0055]

[0037] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a FR1, a FR2, a FR3, and a FR4. The amino acid residue at position 44 of F2 can be a glutamic acid or a glycine, the amino acid at position 45 of F2 can be a arginine or a leucine, the amino acid at position

[0078] of F3 can be a threonine or a serine, and the amino acid at position 79 can be F3 is a leucine or a valine. The FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

[0056]

[0038] The sdAbs disclosed herein can further comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 31, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO:33, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0057]

[0039] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0058]

[0040] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0059]

[0041] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 7

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[0061] 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0062]

[0042] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0063]

[0043] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0064]

[0044] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0065]

[0045] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0066]

[0046] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0067] 8

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[0069]

[0047] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0070]

[0048] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0071]

[0049] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0072]

[0050] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0073]

[0051] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0074]

[0052] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid 9

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[0076] sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0077]

[0053] The sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290 or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290.

[0078]

[0054] The sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42, 44, 49, 53, 55, 56, 57, 6366, 68, 73, 75, 286, 287, 288, 289, or 290, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42, 44, 49, 53, 55, 56, 57, 6366, 68, 73, 75, 286, 287, 288, or 290. Preferably, the sdAb does not comprise the amino acid sequence of SEQ ID NO: 59.

[0079]

[0055] The sdAb can comprise the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 39.

[0080]

[0056] The sdAb can comprise the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 40.

[0081]

[0057] The sdAb can comprise the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 42.

[0082]

[0058] The sdAb can comprise the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 49.

[0083]

[0059] The sdAb can comprise the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 53.

[0084]

[0060] The sdAb can comprise the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 55.

[0085]

[0061] The sdAb can comprise the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 56.

[0086]

[0062] The sdAb can comprise the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 57.

[0087]

[0063] The sdAb does not comprise the amino acid sequence of SEQ ID NO: 59.

[0088]

[0064] The sdAb can comprise the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 63.

[0089] 10

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[0091]

[0065] The sdAb can comprise the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 66.

[0092]

[0066] The sdAb can comprise the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 68.

[0093]

[0067] The sdAb can comprise the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 73.

[0094]

[0068] The sdAb can comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 75.

[0095]

[0069] The sdAb can comprise the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 76.

[0096]

[0070] The sdAb can comprise the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 77.

[0097]

[0071] The sdAb can comprise the amino acid sequence of SEQ ID NO: 286, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 286.

[0098]

[0072] The sdAb can comprise the amino acid sequence of SEQ ID NO: 287, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 287.

[0099]

[0073] The sdAb can comprise the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 288.

[0100]

[0074] The sdAb can comprise the amino acid sequence of SEQ ID NO: 289, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 289.

[0101]

[0075] The sdAb can comprise the amino acid sequence of SEQ ID NO: 290, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 290.

[0102]

[0076] The sdAbs disclosed herein can comprise a half-life of at least about 5 hours to about 40 hours.

[0103]

[0077] The disclosure further relates to pharmaceutical compositions comprising the sdAbs disclosed herein or nucleic acids encoding the sdAbs.

[0104]

[0078] The disclosure further relates to a nucleic acid encoding the sdAbs disclosed herein. The nucleic acid encoding sdAbs can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid of a sdAb.

[0105]

[0079] This disclosure relates to a polypeptide comprising a single domain antibody fragment (sdAb) that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3 and an amino acid encoding a polypeptide with biological activity. The sdAb can have a CDR1 that comprises 11

[0106] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0107] any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions. The sdAb can have a CDR2 that comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. The sdAb can have a CDR3 that comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

[0108]

[0080] The disclosure further relates to a fusion polypeptide comprising a sdAb as disclosed herein and an amino acid sequence of interest. The fusion polypeptide can further comprise an optionally cleavable linker. The optionally cleavable linker can be operably linked to the sdAb and the amino acid sequence of interest. The optionally cleavable linker can be operably linked to the N-terminus of the sdAb. The optionally cleavable linker can be operably linked to the C-terminus of the sdAb. The optionally cleavable linker is preferably cleavable. The optionally cleavable linker can comprise a cleavable moiety that is a substrate for one or more proteases. In some instances, the optionally cleavable linker is not cleavable.

[0109]

[0081] The amino acid sequence of interest can be a polypeptide with biological activity. Exemplary polypeptides with biological activity include, but are not limited to, a cytokine, with the proviso that the cytokine is not IL-2 and / or IL-12 and / or IL-21, a chemokine, a growth factor, a soluble receptor, a tumor antigen binding domain, a peptide hormone, a hormone receptor agonist, or muteins, variants or combinations of any of the foregoing. The cytokine can be IL-10, IL-12, IL-15, IL-18, IL-23, IL-36, IFN, IL-7, Tumor Necrosis Factor (TNF), Granulocyte macrophage colony-stimulating factor (GM-CSF), or a functional fragment or mutein of any of the foregoing. Preferably, the cytokine is not IL-2, IL-12 and / or IL-21. Preferably, the cytokine is not IL-2. Preferably, the cytokine is not IL-12. The chemokine can be CXCL10, CCL19, CCL20, CCL21, or functional fragment of any of the foregoing.

[0110]

[0082] The tumor antigen binding domain can bind to binds EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA.5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, IR WT1.1.

[0111]

[0083] The protease cleavable linker can comprises a sequence that is capable of being cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a 12

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[0113] caspase, a tryptase, or a tumor protease. The protease can be selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G. The protease can be selected from matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14. The protease cleavable linker can comprise at least two sequences that are independently capable of being cleaved by a protease. The protease cleavable linker can comprise a synthetic sequence. The protease cleavable linker can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 214-239 or an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 214-239. The protease cleavable linker can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs.: 214-239. Preferably the protease cleavable linker comprises an amino acid sequence of SEQ ID NO: 217 or an amino acid sequence of SEQ ID NO: 214.

[0114]

[0084] The polypeptide can further comprise an immune cell binding domain. The immune cell binding domain can have specificity for an antigen on a T-cell. The antigen on the T-cell can be CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta.

[0115]

[0085] The polypeptide with biological activity can be a bispecific engager. The bispecific engager can comprise a first binding region with specificity for an antigen on a T cell and a second binding region with specificity for a tumor associated antigen.

[0116]

[0086] The disclosure further relates to a nucleic acid encoding the fusion polypeptide. The nucleic acid composition encoding fusion polypeptide can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid composition of an fusion polypeptide.

[0117]

[0087] The disclosure also relates to a pharmaceutical composition of the fusion polypeptide and methods of making pharmaceutical compositions of the fusion polypeptide. The disclosure further relates to methods for treating a disease, such as cancer, that comprise administering to a subject in need thereof an effective amount of the fusion polypeptide, a nucleic acid encoding the fusion polypeptide, an expression vector for the fusion polypeptide, or a pharmaceutical composition comprising an effective amount of any of these.

[0118]

[0088] This disclosure relates to inducible cytokine prodrugs. The inducible cytokine prodrugs include a sdAb or non-immunoglobulin format that binds HSA, to extend the half-life of the inducible cytokine prodrug as described herein, a cytokine polypeptide and a protease cleavable linker. Typically, the inducible cytokine prodrug further comprises a moiety or domain that binds the cytokine polypeptide (i.e., a blocking domain) and attenuates cytokine activity. The sdAb can also contribute to attenuation, for 13

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[0120] example through steric effects. The blocking domain is capable of blocking all or some of the receptor agonist activity of the cytokine by noncovalently binding to the cytokine (e.g., to IL-2, IL-12, or IFN) and / or sterically blocking receptor binding, for example. Upon cleavage of the protease cleavable linker a form of the cytokine is released that is active (e.g., more active than the inducible cytokine prodrug). Typically, the released cytokine is at least 10 x more active than the cytokine polypeptide prodrug.

[0121] Preferably, the released cytokine is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible cytokine prodrug.

[0122]

[0089] The form of cytokine that is released upon cleavage of the inducible cytokine prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring cytokine. Even though the half-life of the inducible cytokine prodrug is extended, toxicity is reduced or eliminated because the agonist activity of the circulating inducible cytokine prodrug is attenuated, and active cytokine is targeted to the desired site of activity (e.g., tumor microenvironment). The inducible cytokine prodrugs of this disclosure overcome the toxicity and short half-life problems that have severely limited the clinical use pf cytokines in oncology.

[0123]

[0090] The inducible cytokine prodrugs disclosed herein can comprise at least one of each: a) a cytokine polypeptide [A]; b) a sdAb [H] that has binding specificity for human serum albumin; c) a protease cleavable linker [L]; and d) optionally a domain that binds the cytokine polypeptide and inhibits the receptor activating activity of the cytokine polypeptide [D].

[0124]

[0091] The disclosure further relates to an inducible cytokine prodrug comprising at least one of each: a) a cytokine polypeptide [A] with the proviso that the cytokine polypeptide is not IL-21; b) a single domain antibody fragment (sdAb) [H] that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3; c) a protease cleavable linker [L]; and d) optionally a cytokine blocking domain that binds the cytokine polypeptide and inhibits the receptor activating activity of the cytokine polypeptide [D].

[0125]

[0092] The sdAb comprises a CDR1, CDR2 and CDR3. The CDR1 can comprise any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. The CDR2 can comprise any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. The CDR3 can comprise any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

[0126] 14

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[0128]

[0093] The sdAb can function as a half-life extension element, a cytokine blocking element, or both a half-life extension element and a blocking element. Preferably, the sdAb is a half-life extension element. The sdAb can be a cytokine blocking element that also extends in vivo half-life. The sdAb can be a blocking domain that sterically inhibits or blocks activation and / or binding of the cytokine polypeptide to its cognate receptor.

[0129]

[0094] The inducible cytokine prodrug can further comprise a cytokine blocking domain when the sdAb is a half-life extension element.

[0130]

[0095] The cytokine blocking domain can comprise a ligand-binding domain or fragment of a cognate receptor for the cytokine polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the cytokine polypeptide. The antibody or antigen-binding fragment can be a single domain antibody, a Fab, or a scFv that binds the cytokine polypeptide. The cytokine blocking domain can inhibit activation of the cytokine polypeptide receptor by the inducible cytokine prodrug.

[0131]

[0096] The cytokine polypeptide can be IL-2, IL-10, IL-12, IL-15, IL-18, IL-10, IL-23, IL-36, IFNalpha, IFNbeta, IFNgamma, IL-7, Tumor Necrosis Factor (TNF), Granulocyte macrophage colony-stimulating factor (GM-CSF), or a functional fragment or mutein of any of the foregoing. Preferably, the cytokine polypeptide is IL-2 or IL-12. Preferably, the cytokine polypeptide is not IL-21.

[0132]

[0097] The protease cleavable linker can comprise a sequence that is capable of being cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a caspase, a tryptase, or a tumor protease. The protease can be selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G. The protease can be selected from matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14. The protease cleavable linker can comprise at least two sequences that are independently capable of being cleaved by a protease. The protease cleavable linker can comprise a synthetic sequence. The protease cleavable linker can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 214-239 or an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 214-239. Preferably, the protease cleavable linker comprises an amino acid sequence of SEQ ID NO: 214 or SEQ ID NO: 217.

[0133]

[0098] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 amino acid substitutions; and a

[0134] 15

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[0136] CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 amino acid substitutions.

[0137]

[0099] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions.

[0138]

[0100] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0139]

[0101] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0140]

[0102] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0141]

[0103] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0142]

[0104] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0143] 16

[0144] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0145]

[0105] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0146]

[0106] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0147]

[0107] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0148]

[0108] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0149]

[0109] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0150]

[0110] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0151]

[0111] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid 17

[0152] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0153] sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0154]

[0112] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 7; a CDR2 that consists of SEQ ID NO: 38; and a CDR3 that consists of SEQ ID NO: 12.

[0155]

[0113] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 37; a CDR2 that consists of SEQ ID NO: 9; and a CDR3 that consists of SEQ ID NO: 12.

[0156]

[0114] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 10; and a CDR3 that consists of SEQ ID NO: 12.

[0157]

[0115] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 11; and a CDR3 that consists of SEQ ID NO: 12.

[0158]

[0116] The sdAb can comprise CDR1 that consists of SEQ ID NO: 1; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 3.

[0159]

[0117] The sdAb can comprise CDR1 that consists of SEQ ID NO: 4; a CDR2 that consists of SEQ ID NO: 5; and a CDR3 that consists of SEQ ID NO: 6.

[0160]

[0118] The sdAbs disclosed herein can compete with neonatal Fc receptor (FcRn) for binding to HSA. The sdAbs can have binding specificity for domain one of human serum albumin.

[0161]

[0119] The sdAb can be a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain. VH and VHH are preferred sdAbs.

[0162]

[0120] Typically, the sdAb is camelid, human, or humanized. Preferably, the sdAb is humanized.

[0163]

[0121] The sdAb’s disclosed herein further comprise one or more framework regions. The framework region can be derived from a human germline.

[0164]

[0122] The sdAbs disclosed herein can further comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 31, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO:33, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions;

[0165] 18

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[0167] and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0168]

[0123] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0169]

[0124] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0170]

[0125] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0171]

[0126] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0172]

[0127] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0173] 19

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[0175]

[0128] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0176]

[0129] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0177]

[0130] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0178]

[0131] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0179]

[0132] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0180]

[0133] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid 20

[0181] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0182] sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0183]

[0134] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0184]

[0135] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0185]

[0136] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 amino acid substitutions.

[0186]

[0137] The sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 39-79, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39-79.

[0187]

[0138] The sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42, 44, 49, 53, 55, 56, 57, 59, 6366, 68, 73, 75, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39, 40, 42, 44, 49, 53, 55, 56, 57, 59, 6366, 68, 73, 75.

[0188]

[0139] The sdAb can comprise the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 39.

[0189]

[0140] The sdAb can comprise the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 40.

[0190] 21

[0191] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0192]

[0141] The sdAb can comprise the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 42.

[0193]

[0142] The sdAb can comprise the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 49.

[0194]

[0143] The sdAb can comprise the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 53.

[0195]

[0144] The sdAb can comprise the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 55.

[0196]

[0145] The sdAb can comprise the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 56.

[0197]

[0146] The sdAb can comprise the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 57.

[0198]

[0147] The sdAb can comprise the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 59.

[0199]

[0148] The sdAb can comprise the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 63.

[0200]

[0149] The sdAb can comprise the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 66.

[0201]

[0150] The sdAb can comprise the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 68.

[0202]

[0151] The sdAb can comprise the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 73.

[0203]

[0152] The sdAb can comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 75.

[0204]

[0153] The sdAb can comprise the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 76.

[0205]

[0154] The sdAb can comprise the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 77.

[0206]

[0155] The disclosure further relates to a nucleic acid encoding the inducible cytokine prodrugs disclosed herein. The nucleic acid encoding inducible cytokine prodrugs can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid of a sdAb.

[0207] 22

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[0209]

[0156] The disclosure also relates to a pharmaceutical composition of the inducible cytokine prodrug and methods of making pharmaceutical compositions of the fusion polypeptide. The disclosure further relates to methods for treating a disease, such as cancer, that comprise administering to a subject in need thereof an effective amount of the inducible cytokine prodrug, a nucleic acid encoding the inducible cytokine prodrug, an expression vector for the fusion polypeptide, or a pharmaceutical composition comprising an effective amount of any of these.

[0210]

[0157] This disclosure relates to inducible IL-2 prodrugs and / or inducible IL-12 prodrugs.

[0211]

[0158] The inducible IL-2 prodrug can comprise a first polypeptide chain having the formula: [A]-[L1]-[H]-[L2]-[D], [A] is an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 474 or a variant thereof, [L1] is a protease cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 217 or a variant thereof, [H] is a half-life extension element comprising the amino acid sequence of any one of SEQ ID NOs: 480, 39-79, 286-290 or a variant of any of the foregoing, [L2] is a protease cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 217 or a variant thereof, and [D] is an antigen binding portion of a heavy chain comprising the amino acid sequence of SEQ ID NO: 475 or a variant thereof; and a second polypeptide chain comprising an antibody light chain of SEQ ID NO: 476 or a variant thereof that is complementary to the heavy chain in the first polypeptide and together with the light chain forms an IL-2 binding site.

[0212]

[0159] The half-life extension element in the inducible IL-2 prodrug can comprise any one of SEQ ID NOS: 480, 39-79, or 286-290 or an amino acid sequence that is at least about 80% identical to any one of SEQ ID NOs: 480, 39-79, or 286-290.

[0213]

[0160] The half-life extension element in the inducible IL-2 prodrug can be selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, and SEQ ID NO: 290.

[0214]

[0161] The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 39. The half-life element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 40.

[0215] 23

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[0217]

[0162] The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 42. The half-life extension element in the inducible IL-2 prodrug can comprises the amino acid sequence of SEQ ID NO: 43. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 44. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 49. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 53. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 55. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 56. The half-life extension element in the inducible IL-2 prodrug can comprises the amino acid sequence of SEQ ID NO: 57. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 59. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 63. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 66. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 68. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 73. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 75. The half-life extension element in the inducible IL-2 prodrug can comprise the amino acid sequence of SEQ ID NO: 76. The half-life extension element in the inducible IL-2 prodrug an comprise the amino acid sequence of SEQ ID NO: 77.

[0218]

[0163] The inducible IL-2 prodrug can comprise Compound 1 (SEQ ID NO: 326 / SEQ ID NO: 343), Compound 2 (SEQ ID NO: 327 / SEQ ID NO: 343), Compound 3 (SEQ ID NO: 328 / SEQ ID NO: 343), Compound 4 (SEQ ID NO: 329 / SEQ ID NO: 343), Compound 5 (SEQ ID NO: 330 / SEQ ID NO: 343), Compound 6 (SEQ ID NO: 331 / SEQ ID NO: 343), Compound 7 (SEQ ID NO: 332 / SEQ ID NO: 343), Compound 8 (SEQ ID NO: 333 / SEQ ID NO: 343), Compound 9 (SEQ ID NO: 334 / SEQ ID NO: 343), Compound 10 (SEQ ID NO: 335 / SEQ ID NO: 343), Compound 11 (SEQ ID NO: 336 / SEQ ID NO: 343), Compound 12 (SEQ ID NO: 337 / SEQ ID NO: 343), Compound 13 (SEQ ID NO: 338 / SEQ ID NO: 343), Compound 14 (SEQ ID NO: 339 / SEQ ID NO: 343), Compound 15 (SEQ ID NO: 340 / SEQ ID NO: 343), Compound 16 (SEQ ID NO: 341 / SEQ ID NO: 343), Compound 17 (SEQ ID NO: 342 / SEQ ID NO: 343), Compound 117 (SEQ ID NO: 466 / SEQ ID NO: 343), Compound 118 (SEQ ID NO: 482 / SEQ ID NO: 343), Compound 119 (SEQ ID NO: 483 / SEQ ID NO: 343), Compound 120 (SEQ ID NO: 484 / SEQ ID NO: 343), Compound 121 (SEQ ID NO: 485 / SEQ ID NO: 343), Compound 122 (SEQ ID NO: 486 / SEQ ID NO: 343), Compound 123 (SEQ ID NO: 487 / SEQ ID NO: 343), Compound 124 (SEQ ID NO:

[0219] 488 / SEQ ID NO: 343), Compound 125 (SEQ ID NO: 489 / SEQ ID NO: 343), Compound 126 (SEQ ID 24

[0220] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0221] NO: 490 / SEQ ID NO: 343), Compound 127 (SEQ ID NO: 491 / SEQ ID NO: 343), Compound 128 (SEQ ID NO: 492 / SEQ ID NO: 343), Compound 129 (SEQ ID NO: 493 / SEQ ID NO: 343), Compound 130 (SEQ ID NO: 494 / SEQ ID NO: 343), Compound 131 (SEQ ID NO: 495 / SEQ ID NO: 343), Compound 132 (SEQ ID NO: 496 / SEQ ID NO: 343), Compound 133 (SEQ ID NO: 497 / SEQ ID NO: 343), Compound 134 (SEQ ID NO: 498 / SEQ ID NO: 343), Compound 135 (SEQ ID NO: 499 / SEQ ID NO: 343), Compound 136 (SEQ ID NO: 500 / SEQ ID NO: 343), Compound 137 (SEQ ID NO: 501 / SEQ ID NO: 343), Compound 138 (SEQ ID NO: 502 / SEQ ID NO: 343), Compound 139 (SEQ ID NO: 503 / SEQ ID NO: 343), Compound 140 (SEQ ID NO: 504 / SEQ ID NO: 343), Compound 141 (SEQ ID NO: 505 / SEQ ID NO: 343), Compound 142 (SEQ ID NO: 506 / SEQ ID NO: 343), Compound 143 (SEQ ID NO: 507 / SEQ ID NO: 343), Compound 144 (SEQ ID NO: 508 / SEQ ID NO: 343), Compound 145 (SEQ ID NO: 509 / SEQ ID NO: 343), and Compound 146 (SEQ ID NO: 510 / SEQ ID NO: 343) or a variant of the foregoing.

[0222] The inducible IL-2 prodrug can comprise: a first polypeptide chain that comprises an amino acid sequences selected from a) SEQ ID NO: 326 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 326, b) SEQ ID NO: 327 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 327, c) SEQ ID NO: 328 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 328, d) SEQ ID NO: 329 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 329, e) SEQ ID NO: 330 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 330, f) SEQ ID NO: 331 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 331, g) SEQ ID NO: 332 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 332, h) SEQ ID NO: 333 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 333, i) SEQ ID NO: 334 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 334, j) SEQ ID NO: 335 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 335, k) SEQ ID NO: 336 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 336, l) SEQ ID NO: 337 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 337, m) SEQ ID NO: 338 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 338, n) SEQ ID NO: 339 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 339, o) SEQ ID NO: 340 a or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 340, p) SEQ ID NO: 341 a or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 341, and q) SEQ ID NO: 342 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 342; a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 343 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 343.

[0223] 25

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[0225]

[0164] The inducible IL-12 prodrug can comprise a first polypeptide having the formula: [H]-[L1]-[A]-[L2]-[D], wherein [H] is a half-life extension element comprising the amino acid sequence of any one of SEQ ID NOs: 39-79 and 286-290, or a variant of any of the foregoing, [L1] is a protease cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 217 or a variant thereof, [A] is a p35 subunit comprising the amino acid sequence of SEQ ID NO: 477 or a variant thereof, [L2] is a protease cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 217 or a variant thereof, and [D] is a single chain variable fragment comprising the amino acid sequence of SEQ ID NO: 478 or a variant thereof, and a second polypeptide chain comprising a p40 subunit comprising the amino acid sequence of SEQ ID NO: 444.

[0226]

[0165] The half-life extension element in the inducible IL-12 prodrug can comprise any one of SEQ ID NOS: 39-79 or 286-290 or an amino acid sequence that is at least about 80% identical to any one of SEQ ID NOs.: 39-79 or 286-290.

[0227]

[0166] The half-life extension element in the inducible IL-12 prodrug can be selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, and SEQ ID NO: 290.

[0228]

[0167] The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 39. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 40. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 42. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 43. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 44. The half-life extension element in the inducible IL-12 prodrug comprises the amino acid sequence of SEQ ID NO: 49. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 53. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 55. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 56. The half-life extension element in 26

[0229] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0230] the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 57. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 59. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 63. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 66. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 68. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 73. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 75. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 76. The half-life extension element in the inducible IL-12 prodrug can comprise the amino acid sequence of SEQ ID NO: 77.

[0231]

[0168] The inducible IL-12 prodrug can comprise Compound 148 (SEQ ID NO: 512 / SEQ ID NO: 444), Compound 149 (SEQ ID NO: 513 / SEQ ID NO: 444), Compound 150 (SEQ ID NO: 514 / SEQ ID NO: 444), Compound 151 (SEQ ID NO: 515 / SEQ ID NO: 444), Compound 152 (SEQ ID NO: 516 / SEQ ID NO: 444), Compound 153 (SEQ ID NO: 517 / SEQ ID NO: 444), Compound 154 (SEQ ID NO: 518 / SEQ ID NO: 444), Compound 155 (SEQ ID NO: 519 / SEQ ID NO: 444), Compound 156 (SEQ ID NO:

[0232] 520 / SEQ ID NO: 444), Compound 157 (SEQ ID NO: 521 / SEQ ID NO: 444), Compound 158 (SEQ ID NO: 522 / SEQ ID NO: 444), Compound 159 (SEQ ID NO: 523 / SEQ ID NO: 444), Compound 160 (SEQ ID NO: 524 / SEQ ID NO: 444), Compound 161 (SEQ ID NO: 525 / SEQ ID NO: 444), Compound 162 (SEQ ID NO: 526 / SEQ ID NO: 444), Compound 163 (SEQ ID NO: 527 / SEQ ID NO: 444), Compound 164 (SEQ ID NO: 528 / SEQ ID NO: 444), Compound 165 (SEQ ID NO: 529 / SEQ ID NO: 444), Compound 166 (SEQ ID NO: 530 / SEQ ID NO: 444), Compound 167 (SEQ ID NO: 531 / SEQ ID NO: 444), Compound 168 (SEQ ID NO: 532 / SEQ ID NO: 444), Compound 169 (SEQ ID NO: 533 / SEQ ID NO: 444), Compound 170 (SEQ ID NO: 534 / SEQ ID NO: 444), Compound 171 (SEQ ID NO: 535 / SEQ ID NO: 444), Compound 172 (SEQ ID NO: 536 / SEQ ID NO: 444), Compound 173 (SEQ ID NO:

[0233] 537 / SEQ ID NO: 444), Compound 174 (SEQ ID NO: 538 / SEQ ID NO: 444), Compound 175 (SEQ ID NO: 539 / SEQ ID NO: 444), Compound 176 (SEQ ID NO: 540 / SEQ ID NO: 444), Compound 177 (SEQ ID NO: 541 / SEQ ID NO: 444), Compound 178 (SEQ ID NO: 542 / SEQ ID NO: 444), Compound 179 (SEQ ID NO: 543 / SEQ ID NO: 444), Compound 180 (SEQ ID NO: 544 / SEQ ID NO: 444), Compound 181 (SEQ ID NO: 545 / SEQ ID NO: 444), Compound 182 (SEQ ID NO: 546 / SEQ ID NO: 444), Compound 183 (SEQ ID NO: 547 / SEQ ID NO: 444), Compound 184 (SEQ ID NO: 548 / SEQ ID NO: 444), Compound 185 (SEQ ID NO: 549 / SEQ ID NO: 444), Compound 186 (SEQ ID NO: 550 / SEQ ID

[0234] 27

[0235] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0236] NO: 444), Compound 187 (SEQ ID NO: 551 / SEQ ID NO: 444), and Compound 188 (SEQ ID NO:

[0237] 552 / SEQ ID NO: 444), or a variant of any of the foregoing.

[0238]

[0169] The inducible IL-12 prodrug can comprise: a first polypeptide chain that comprises an amino acid sequence selected from a) SEQ ID NO: 512 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 512, b) SEQ ID NO: 519 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 519, c) SEQ ID NO: 521 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 521, d) SEQ ID NO: 526 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 526, e) SEQ ID NO: 530 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 530, f) SEQ ID NO: 516 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 516, g) SEQ ID NO: 532 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 532, h) SEQ ID NO: 513 or an amino acid sequence that has at least about 80% identity to SEQ ID NO:513, i) SEQ ID NO: 514 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 514, j) SEQ ID NO: 515 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 515, k) SEQ ID NO: 539 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 539, l) SEQ ID NO: 540 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 540, m) SEQ ID NO: 546 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 546, n) SEQ ID NO: 548 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 548, o) SEQ ID NO: 549 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 549, and p) SEQ ID NO: 550 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 550; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 479 or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 444

[0239]

[0170] This disclosure further relates to methods and compositions for treating cancer using a combination therapy comprising an inducible IL-12 prodrug in combination with an inducible IL-2 prodrug.

[0240]

[0171] The disclosure further relates to nucleic acid encoding the inducible IL-2 prodrugs and / or inducible IL-12 prodrugs. The nucleic acid encoding inducible IL-2 prodrugs and / or IL-12 prodrugs can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid of a inducible IL-2 prodrug and / or an inducible IL-12 prodrug.

[0241]

[0172] The disclosure also relates to a pharmaceutical composition of the inducible IL-2 prodrugs and / or inducible IL-12 prodrugs and methods of making pharmaceutical compositions of the inducible IL-2

[0242] 28

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[0244] prodrugs and / or inducible IL-12 prodrugs. The disclosure further relates to methods for treating a disease, such as cancer.

[0245] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0246]

[0173] The drawings are not necessarily to scale or exhaustive. Instead, the emphasis is generally placed upon illustrating the principles of the inventions described herein. The accompanying drawings, which constitute a part of the specification, illustrate several embodiments consistent with the disclosure and,together with the description, serve to explain the principles of the disclosure. In the drawings:

[0247]

[0174] FIGs.1A-1H are images of SDS-PAGE gels showing anti-human serum albumin VHH antibodies derived from llamas. The VHHs were 6xHis-tagged (SEQ ID NO: 570) (FIGs.1A-1C), Fc-tagged (FIGs.1D-1G), or 6xHis- (SEQ ID NO: 570) and AviTagged (FIG.1H). NR refers to non-reduced conditions, and R refers to reduced conditions.

[0248]

[0175] FIGs.2A-2J are tables showing sequence alignments of humanized variants of llama anti-human serum albumin VHH antibodies. Alignments of all anti-HSA VHHs is shown in FIG.2A (SEQ ID NOS 565 and 80-149, respectively, in order of appearance). Alignments of top CDR3 Group 1 cross reactive VHHs is shown in FIG.2B (SEQ ID NOS 566, 80-91, 97, 99, 102 and 106, respectively, in order of appearance). Alignments of CDR3 Group 5 cross reactive VHHs is shown in FIG.2C (SEQ ID NOS 125, 124, 125 and 126, respectively, in order of appearance). Alignments of top CDR1 groups 1 and 5 cross reactive VHH’s. Figure 2D discloses SEQ ID NOS 567, 80-91, 97, 99, 102, 106 and 124-126, respectively, in order of appearance. Alignments of each llama VHH, the chosen human germline Vh template protein(s), and humanized variants are shown in FIG.2E (1HUM19) (SEQ ID NOS 40, 568, 569 and 39-47, respectively, in order of appearance), FIG.2F (3HUM181) (SEQ ID NOS 571, 572, 569 and 56-65, respectively, in order of appearance), FIG.2G (2HUM45 on a IGHV3-23 backbone) (SEQ ID NOS 69, 573, 574 and 66-70, respectively, in order of appearance), FIG.2H, (2HUM45 on an IGHV3-11 backbone) (SEQ ID NOS 575, 573, 576 and 71-79, respectively, in order of appearance), FIG.2I (1HUM19 variants with modified CDR sequences) (SEQ ID NOS 577, 568, 569, 48, 50, 50-55, 564, 564, 286 and 578, respectively, in order of appearance), and FIG.2J (3HUM181 variants with modified CDR sequences) (SEQ ID NOS 579-582 and 289, respectively, in order of appearance). The CDRs are indicated.

[0249]

[0176] FIGs.3A-3J are images of SDS-PAGE gels showing humanized anti-human serum albumin VHH antibodies. The humanized VHH antibodies were run under non-reduced conditions (FIGs.3A-3E) and under reduced and non-reduced conditions (FIGs.3F-3J).

[0250] 29

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[0252]

[0177] FIGs.4A and 4B are graphs showing predicted PK (µg / mL) over time of anti-albumin VHHs with varying binding affinities to mouse (FIG.4A) or human serum albumin (FIG.4B).

[0253]

[0178] FIGs.5A-5D are images of SDS-PAGE gels showing inducible IL-2 prodrugs comprising an anti-human serum albumin VHH antibody derived from llama (WW10321 / WW00523, WW10322 / WW00523, WW10323 / WW00523, WW10324 / WW00523). NR refers to non-reduced conditions, and R refers to reduced conditions.

[0254]

[0179] FIG.6 is a graph of the showing plasma concentration profiles for inducible IL-2 prodrugs comprising an anti-human serum albumin VHH antibody derived from llama (WW10321 / WW00523, WW10322 / WW00523, WW10323 / WW00523, WW10324 / WW00523) over time. The graph shows that the concentration for each of the VHH-IL-2 inducible prodrug followed a similar, non-linear pharmacokinetic (PK) profile, with a steep terminal elimination phase.

[0255]

[0180] FIGs.7A-7F are graphs showing results of analyzing inducible IL-2 prodrug variants in a syngeneic MC38 mouse tumor model. Tumor volume over time in mice treated with vehicle or 25 µg, 50 µg, or 100 µg of WW00621 / WW00523 (FIG.7A), (WW10405 / WW00523) (FIG.7B), (WW10409 / WW00523) (FIG.7C), (WW10410 / WW00523) (FIG.7D), (WW10414 / WW00523) (FIG.

[0256] 7E), or (WW10420 / WW00523) (FIG.7F) are shown.

[0257]

[0181] FIGs.8A-8F show the average body weight of the animals treated in the experiment shown in FIGs.7A-7F. No significant loss in body weight was observed in any of the dose levels across all tested inducible IL-2 prodrugs.

[0258]

[0182] FIGs.9A-9C are graphs showing tumor volume over time in mice treated in the experiment shown in FIGs 7A-7F with vehicle, or 25 µg (FIG.9A), 50 µg (FIG.9B), or 100 µg (FIG.9C) of WW00621 / WW00523, (WW10405 / WW00523), (WW10409 / WW00523), (WW10410 / WW00523), (WW10414 / WW00523), or (WW10420 / WW00523).

[0259]

[0183] FIGs.10A-10S are graphs showing tumor volume over time for each individual animal treated in the experiment shown in FIGs.9A-9C.

[0260]

[0184] FIGs.11A-11C are graphs showing the anti-tumor activity of WW0757 / 00636 (an inducible IL-12 prodrug comprising a chimeric IL-12 polypeptide and albumin binding half extension domain, 10GE) (FIG.11A), WW10465 / 00636 (an inducible IL-12 prodrug comprising a half-life extension domain with a comparable albumin binding affinity to 10GE) (FIG.11B), and WW10545 / 00636 (an inducible IL-12 prodrug comprising a half-life extension domain with a lower albumin binding affinity than 10GE) (FIG.

[0261] 11C). It shows average tumor volume over time in mice treated. Vehicle treated animals are shown as

[0262] 30

[0263] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0264] black circles. The data show that the tumor growth inhibition by both WW10465 / 00636 and WW10545 / 00636 was comparable to WW0757 / 00636 at equivalent doses.

[0265]

[0185] FIGs.12A-12C show percent body weight loss after dosing with fusion proteins in the MC38 mouse model corresponding to the data shown in FIGs.11A-11C.

[0266]

[0186] FIG.13 is a graph the peripheral concentrations of the inducible IL-12 prodrugs over time. The data show that the exposure of WW10465 / 00636 is comparable to that of WW0757 / 636, whereas the half-life of WW10545 / 00636 is significantly shorter than WW0757 / 00636.

[0267]

[0187] FIGs.14A, 14C, 14E, 14G, 14I, 14K, 14M, and 14O are a series of graphs showing activity of conditionally activated IL-2 comprising a humanized anti-HSA VHH in an IL-2 luciferase reporter assay.

[0268] FIGs.14B, 14D, 14F, 14H, 14J, 14L, 14N, 14P are images of SDS-PAGE gels showing the results of protein cleavage assays using the conditionally activated IL-2 used in the assays shown in FIGs.14A, 14C, 14E, 14G, 14I, 14K, 14M, and 14O, respectively. Closed squares depict activity of the uncut IL-2 prodrug (intact) and open squares depict the activity of the cut form of the IL-2 prodrug (cleaved). Circles depict the positive control (recombinant human IL-2) and diamonds depict the negative control (cells only). EC50 values for each are shown in the table. Constructs tested and depicted include WW10405 / WW00523 (FIGs.14A and 14B), WW10409 / WW00523 (FIGs.14C and 14D), WW10410 / WW00523 (FIGs.14E and 14F), WW10413 / WW00523 (FIGs.14G and 14H), WW10414 / WW00523 (FIGs.14I and 14J), WW10420 / WW00523 (FIGs.14K and 14L), WW10416 / WW00523 (FIGs.14M and 14N), WW10417 / WW00523 (FIGs.14O and 14P).

[0269] 4. DETAILED DESCRIPTION

[0270]

[0188] The disclosure relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof. Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein. Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.

[0271]

[0189] As described and exemplified herein, contrary to the well-accepted knowledge in the art that anti-albumin binding domains that interfere with albumin binding to FcRn are not suitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short half-life therapeutic proteins), the inventors have discovered that sdAbs that compete with FcRn (human FcRn) for binding to

[0272] 31

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[0274] human serum albumin can effectively extend the half-life (e.g., the in vivo serum half-life) of short half-life therapeutic agents (e.g., proteins, peptides).

[0275] A. sdAbs that bind Serum Albumin

[0276]

[0190] The sdAbs of this disclosure include, but are not limited to, heavy chain variable domain (VH), variable heavy domain of heavy chain (VHH), single domain shark variable domain of new antigen receptor (VNAR), or light chain variable (VL) domain (e.g., a kappa VL domain or a lambda VL domain).

[0277]

[0191] The sdAbs comprise one or more complementary determining regions (CDRs), typically three CDRs, of an immunoglobulin single variable domain that has binding specificity for serum albumin. The CDRs of the sdAbs disclosed herein can be derived from a camelid. The sdAbs can be humanized and comprise one or more FRs, that can be derived from a human germline and typically include back mutations. The sdAbs disclosed herein can comprise a CDR1, a CDR2 and / or a CDR3. Preferably, the sdAbs disclosed herein comprise CDR1, CDR2, and CDR3.

[0278]

[0192] The sdAbs can comprise a CDR1 comprising an amino acid sequence of Formula (I):

[0279] X1X2X3X4X5X6AX7X8G (I).

[0280]

[0193] In Formula (I), X1is glycine or aspartic acid, X2is glycine, arginine, phenylalanine, leucine, valine, serine, tyrosine, histidine, or threonine, X3is threonine, glycine, or serine, X4is phenylalanine, valine, tyrosine, leucine, isoleucine, or arginine, X5is aspartic acid, serine, threonine, or arginine, X6is aspartic acid, serine, glycine, or glutamic acid, X7is arginine, glycine, serine, threonine, methionine, valine, isoleucine, histidine, or asparagine and X8is arginine, glycine, leucine, methionine, isoleucine or valine.

[0281]

[0194] The sdAbs can comprise a CDR2 comprising an amino acid of Formula (II)

[0282] A I S X1S X2X3X4T X5Y X6X7X8V K G (II) (SEQ ID NO: 561)

[0283]

[0195] In Formulas (II), X1is isoleucine or alanine, X2is glycine or threonine, X3is glutamic acid or glycine, X4is arginine, threonine, or serine, X5is asparagine, arginine, or tyrosine, X6is glycine, arginine, serine, or glutamic acid, X7is glutamic acid, lysine, aspartic acid, or glutamic acid, and X8is serine or glutamic acid.

[0284]

[0196] The sdAbs can comprise a CDR3 comprising an amino acid sequence of Formula (III):

[0285] A X1G D W Y H L X2Q X3X4X5X6X7X8I X9X10(III) (SEQ ID NO: 562).

[0286]

[0197] In Formula (III), X1is serine or alanine, X2is valine, isoleucine, or threonine, X3is glycine or glutamic acid, X4is threonine, glutamic acid, or histidine, X4is threonine, glutamic acid, or histidine, X5is 32

[0287] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0288] glycine or asparagine, X6us methionine or asparagine, X7is serine, alanine, or methionine, X8is tyrosine or phenylalanine, X9is arginine or tryptophan, and X10is asparagine or tyrosine.

[0289]

[0198] In embodiments, the sdAbs, comprises a CDR1 of Formula (I), a CDR2 of Formula (II), and a CDR3 of Formula (III).

[0290]

[0199] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 1. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 4. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 7. The sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 8. The sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 37.

[0291]

[0200] The sdAb disclosed herein can comprise a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 5, 9, 10, 11, 38 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 2. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 5. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 9. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 10. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 11. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 38.

[0292]

[0201] The sdAbs disclosed herein can comprise a CDR3 as defined by any one of SEQ ID NOs: 3, 6, or 12, or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 3. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 6. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 12.

[0293]

[0202] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of the foregoing, a CDR2 comprising any one of SEQ ID NO: 2, 5, 9, 10, 11, 38, or a variant of any of the foregoing, and a CDR3, such that the sdAbs binds human serum albumin.

[0294]

[0203] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of the foregoing, and a CDR2, such that the sdAbs binds human serum albumin.

[0295] 33

[0296] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0297]

[0204] The sdAbs disclosed herein can comprise a CDR2 comprising any one of SEQ ID NO: 2, 5, 9, 10, 11, 38, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of the foregoing, and a CDR1, such that the sdAbs binds human serum albumin.

[0298]

[0205] Typically, the sdAbs disclosed herein have all three CDRs (e.g., a CDR1, a CDR2, and a CDR3).

[0299]

[0206] The sdAbs disclosed herein can comprise a CDR1 comprising any one of SEQ ID NO: 1, 4, 7, 8 and 37, or a variant of any of the foregoing; a CDR2 comprising any one of SEQ ID NO: 2, 5, 9, 10, 11 and 38 or a variant of any of the foregoing; and a CDR3 comprising any one of SEQ ID NO: 3, 6, and 12, or a variant of any of the foregoing.

[0300]

[0207] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 1 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 2 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 3 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 1, CDR2 that comprises or consists of SEQ ID NO: 2, and CDR3 that comprises or consists of SEQ ID NO: 3.

[0301]

[0208] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 4 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 5 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 6 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 4, CDR2 that comprises or consists of SEQ ID NO: 5, and CDR3 that comprises or consists of SEQ ID NO: 6.

[0302]

[0209] The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO:12 or variant thereof, CDR1 that comprises or consists of SEQ ID NO: 7, 37 or 8 or variant of any of the foregoing, and CDR2 that comprises or consists of SEQ ID NO: 38, 9, 10 or 11 or variant of any of the foregoing. The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO:12, CDR1 that comprises or consists of SEQ ID NO: 7, 37 or 8, and CDR2 that comprises or consists of SEQ ID NO: 38, 9, 10 or 11.

[0303]

[0210] For example, the sdAbs can comprise a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of 34

[0304] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0305] SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID 35

[0306] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0307] NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0308]

[0211] Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 7; a CDR2 that comprises or consists of SEQ ID NO: 38; and a CDR3 that comprises or consists of SEQ ID NO: 12. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 37; a CDR2 that comprises or consists of SEQ ID NO: 9; and a CDR3 that comprises or consists of SEQ ID NO: 12. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 8; a CDR2 that comprises or consists of SEQ ID NO: 10; and a CDR3 that comprises or consists of SEQ ID NO: 12. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 8; a CDR2 that comprises or consists of SEQ ID NO: 11; and a CDR3 that comprises or consists of SEQ ID NO: 12. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 1; a CDR2 that comprises or consists of SEQ ID NO: 2; and a CDR3 that comprises or consists of SEQ ID NO: 3. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 4; a CDR2 that comprises or consists of SEQ ID NO: 5; and a CDR3 that comprises or consists of SEQ ID NO: 6.

[0309]

[0212] The CDRs of the sdAbs disclosed herein are interposed between stretches amino acids referred to as framework regions (FRs). The sdAbs disclosed herein can comprise FR1, FR2, FR3, and / or F4. In certain embodiments, the FRs regions of the sdAbs disclosed herein comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of amino acid sequence homology to human FRs. Typically, the FRs can be derived from a human germline gene. Exemplary human germline genes include, but are not limited to, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-19, IGHV3-20, IGHV3-21, IGHV3-22, IGHV3-23, IGHV3-25, IGHV3-29, IGHV3-30, IGHV3-32, IGHV3-33, IGHV3-35, IGHV3-36, IGHV3-37, IGHV3-38, IGHV3-41, IGHV3-42, IGHV3-43, IGHV3-47, IGHV3-38, IGHV3-49, IGHV3-50, IGHV3-52, IGHV3-53, IGHV3-54, IGHV3-57, IGHV3-60, IGHV3-62, IGHV3-63, IGHV3-64, IGHV3-66, IGHV3-69, IGHV3-71, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-75, IGHV3-76, or IGHV3-79. Certain preferred sdAbs include FRs derived from human germline gene IGHV3-11 and / or IGHV3-23.

[0310]

[0213] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 13, 14, 31, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR1 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs.: SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 13. The sdAbs disclosed herein can include 36

[0311] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0312] a FR1 that comprises or consists of SEQ ID NO: 14. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 31.

[0313]

[0214] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs:15-19, 24-28, 32, 33, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 15. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 16. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 17. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 18. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 19. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 24. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 25. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 26. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 27. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 28. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 32. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 33.

[0314]

[0215] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 20. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 21. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 34. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 35. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 36.

[0315]

[0216] The sdAbs disclosed herein can comprise a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof. In some instances, the sdAbs disclosed herein can comprise a FR4 comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof. The sdAbs disclosed herein can include a FR4 that comprises or consists of SEQ ID NO: 22.

[0316]

[0217] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 37

[0317] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0318] 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing.

[0319]

[0218] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing.

[0320]

[0219] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0321]

[0220] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing.

[0322]

[0221] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0323]

[0222] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0324] 38

[0325] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0326]

[0223] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing.

[0327]

[0224] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0328]

[0225] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0329]

[0226] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino

[0330] 39

[0331] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0332] acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0333]

[0227] Typically, the sdAbs disclosed herein have all four FRs (e.g., a FR1, a FR2, a FR3, and a FR4). The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0334]

[0228] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 15, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0335]

[0229] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 16, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0336]

[0230] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 17, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0337]

[0231] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 18, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0338]

[0232] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 15, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0339]

[0233] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 16, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0340] 40

[0341] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0342]

[0234] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 17, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0343]

[0235] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 18, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0344]

[0236] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 19, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0345]

[0237] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0346]

[0238] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0347]

[0239] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 26, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0348]

[0240] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0349]

[0241] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0350]

[0242] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0351]

[0243] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 26, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0352] 41

[0353] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0354]

[0244] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0355]

[0245] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 28, a FR3 that comprises or consists of SEQ ID NO: 21; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0356]

[0246] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 30; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0357]

[0247] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0358]

[0248] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0359]

[0249] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0360]

[0250] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0361]

[0251] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0362]

[0252] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0363]

[0253] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0364] 42

[0365] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0366]

[0254] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0367]

[0255] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0368]

[0256] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0369]

[0257] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0370]

[0258] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0371]

[0259] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 28, a FR3 that comprises or consists of SEQ ID NO: 36; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0372]

[0260] The sdAbs disclosed herein can comprise or consist of any one of the sdAbs disclosed in Table 1.

[0373] Table 1. Exemplary sdAbs

[0374] sdAb FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 1HUM19A SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID

[0375] D

[0376] D

[0377] D

[0378] D

[0379]

[0380] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0381] 1HUM19_F SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 14 NO: 7 NO: 16 NO: 38 NO: 20 NO: 12 NO: 22 NO:44

[0382] D

[0383] D

[0384] D

[0385] D

[0386] D

[0387] D

[0388] D

[0389] D

[0390] D

[0391] D

[0392] D

[0393] D

[0394] D

[0395] D

[0396] D

[0397]

[0398] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0399] 3HUM181_ SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID F SEQ ID NO: 14 NO: 1 NO: 25 NO: 2 NO: 29 NO: 3 NO: 22 NO: 61

[0400] D

[0401] D

[0402] D

[0403] D

[0404] D

[0405] D

[0406] D

[0407] D

[0408] D

[0409] D

[0410] D

[0411] D

[0412] D

[0413] D

[0414] D

[0415]

[0416] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0417] 2HUM45_L SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID

[0418] SEQ ID NO: 31 NO: 4 NO: 27 NO: 5 NO: 35 NO: 6 NO: 22 NO: 77

[0419] D

[0420] D

[0421]

[0422]

[0261] The sdAbs disclosed herein can comprises or consists of the amino acid sequence of any of SEQ ID NOs: 39-58, 60-79 or 286-290, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 39-59, 60-79 or 286-290. For example, the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 286, 287, 288, 289, or 290. Preferably, the sdAbs do not comprise or consist of the amino acid sequence of SEQ ID NO: 59.

[0423]

[0262] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 39.

[0424]

[0263] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 40.

[0425] 46

[0426] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0427]

[0264] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 41.

[0428]

[0265] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 42.

[0429]

[0266] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 43.

[0430]

[0267] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 44. The sdAb

[0431]

[0268] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 45.

[0432]

[0269] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 46.

[0433]

[0270] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 47.

[0434]

[0271] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 48.

[0435]

[0272] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 49.

[0436]

[0273] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 50.

[0437]

[0274] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 51.

[0438]

[0275] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 52.

[0439]

[0276] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 53.

[0440]

[0277] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 54.

[0441]

[0278] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 55.

[0442]

[0279] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 56.

[0443] 47

[0444] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0445]

[0280] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 57.

[0446]

[0281] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 58.

[0447]

[0282] The sdAbs does not comprise or consist of the amino acid sequence of SEQ ID NO: 59.

[0448]

[0283] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 60.

[0449]

[0284] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 61.

[0450]

[0285] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 62.

[0451]

[0286] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 63.

[0452]

[0287] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 64.

[0453]

[0288] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 65.

[0454]

[0289] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 66.

[0455]

[0290] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 67.

[0456]

[0291] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 68.

[0457]

[0292] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 69.

[0458]

[0293] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 70.

[0459]

[0294] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 71.

[0460]

[0295] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 72.

[0461] 48

[0462] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0463]

[0296] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 73.

[0464]

[0297] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 74.

[0465]

[0298] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 75.

[0466]

[0299] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 76.

[0467]

[0300] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 77.

[0468]

[0301] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 78.

[0469]

[0302] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 79.

[0470]

[0303] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 286, or an amino acid sequence that has at least 80% identity to SEQ UD NO: 286.

[0471]

[0304] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 287, or an amino acid sequence that has at least 80% identity to SEQ UD NO: 287.

[0472]

[0305] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 288, or an amino acid sequence that has at least 80% identity to SEQ UD NO: 288.

[0473]

[0306] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 289, or an amino acid sequence that has at least 80% identity to SEQ UD NO: 289.

[0474]

[0307] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 290, or an amino acid sequence that has at least 80% identity to SEQ UD NO: 290.

[0475] B. sdAbs Sequence Variants

[0476]

[0308] The sdAbs disclosed herein include functional variants that differ in amino acid sequence in comparison, for example, to any one of SEQ ID NOs.: 39-58, 60-79 or 286-290. A “functional variant” of a sdAb retains the ability to bind to serum albumin.

[0477]

[0309] The sdAbs disclosed herein can comprise one or more amino acid substitutions. The amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution. Conservative amino acid substitutions are generally made in accordance with the following Table 2.

[0478] 49

[0479] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0480] Table 2. Exemplary amino acid substitutions

[0481] Amino Acid Exemplary Substitutions

[0482]

[0483] s escre eren, a s o s scosure can conan a C a comprses or consss of SEQ ID NO: 1, 4, 7, 8, or 37 or a variant of any of the forgoing. A variant of SEQ ID NO: 1, 4, 7, 8, or 37 has at least about 80% or at least about 90% amino acid sequence identity to SEQ ID NO: 1, 4, 7, 8, or 37; a variant of SEQ ID NO: 1, 4, 7, 8 or 37 can contain one or two amino acid substitutions. As amino acid substitutions in the CDRs (i.e., CDR1, CDR2 and / or CDR3) can affect binding to serum albumin, it is preferred that any such substitutions are conservative substitutions. One or more amino acids in a CDR can be replaced with a human germ line amino acid, for example, when it is desirable to decrease the binding affinity.

[0484]

[0311] As described herein, a sdAb of this disclosure can contain a CDR2 that comprises or consists of SEQ ID NO: 2, 5, 9, 10, 11 or 38 or a variant of any of the forgoing. A variant of SEQ ID NO: 2, 5, 9, 10, 11 or 38 has at least about 80%, at least about 85%, or at least about 90% amino acid sequence identity to SEQ ID NO: 2, 5, 9, 10, 11 or 38; a variant of SEQ ID NO: 2, 5, 9, 10, 11 or 38 can contain one or two or three amino acid substitutions.

[0485] 50

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[0487]

[0312] As described herein, a sdAb of this disclosure can contain a CDR3 that comprises or consists of SEQ ID NO: 3, 6 or 12 or a variant of any of the forgoing. A variant of SEQ ID NO: 3, 6 or 12 has at least about 80%, at least about 85% or at least about 90% amino acid sequence identity to SEQ ID NO: 3, 6 or 12; a variant of SEQ ID NO: 3, 6 or 12 can contain one or two or three amino acid substitutions.

[0488]

[0313] As described herein, a sdAb of this disclosure can contain FRs and these sequences can generally include amino acid substitutions without causing a loss of binding activity. As described herein, a sdAb of this disclosure can contain a FR1 that comprises or consists of SEQ ID NO: 13, 14, or 31 or a variant of any of the forgoing. A variant of SEQ ID NO: 3, 6 or 12 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 3, 6 or 12.

[0489]

[0314] As described herein, a sdAb of this disclosure can contain a FR2 that comprises or consists of SEQ ID NO: 15-19, 24-28, 32, or 33 or a variant of any of the forgoing. A variant of SEQ ID NO: 15-19, 24-28, 32, or 33 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 15-19, 24-28, 32, or 33.

[0490]

[0315] As described herein, a sdAb of this disclosure can contain a FR3 that comprises or consists of SEQ ID NO: 20, 21, 29, 30, 34, 35, or 36 or a variant of any of the forgoing. A variant of SEQ ID NO: 20, 21, 29, 30, 34, 35, or 36 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 20, 21, 29, 30, 34, 35, or 36.

[0491]

[0316] As described herein, a sdAb of this disclosure can contain a FR4 that comprises or consists of SEQ ID NO: 22 or a variant thereof. A variant of SEQ ID NO: 22 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 22.

[0492]

[0317] The sdAb disclosed herein comprising the amino acid sequence of any one of SEQ ID NO: 39-58, 60-79 or 286-290 can comprises any one of the following amino acids: 1) the amino acid at residue at position 24 can be selected from alanine or serine; 2) the amino acid at position 44 can be selected from glutamic acid, glycine, or aspartic acid; 3) the amino acid at position 45 can be selected from arginine, or leucine; 4) the amino acid at position 78 can be selected from asparagine, serine, threonine, or leucine; or 5) the amino acid at position 79 can be selected from valine, leucine, or phenylalanine.

[0493] C. Properties of Single Anti-Serum Albumin Binding Fragments

[0494]

[0318] Suitable sdAbs disclosed herein can have any one or any combination of the features and properties described in detail herein, including those described in this section.

[0495] 51

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[0497]

[0319] The sdAbs bind human serum albumin (HSA). The sdAbs can additionally bind to serum albumin from one or more additional species such as, a non-human primate (e.g., Rhesus macaque, Cynomolgus macaque and the like) and / or rodent (e.g., mouse, rat and the like).

[0498]

[0320] The sdAbs bind to HSA having the amino acid sequence of SEQ ID NO: 323 or a variant thereof.

[0499]

[0321] The sdAbs preferably compete with FcRn for binding to serum albumin. The sdAbs can bind Domain I or Domain III serum albumin. The sdAbs preferably compete with FcRn for binding to serum albumin and bind Domain I of serum albumin. The sdAbs preferably compete with FcRn for binding to serum albumin and bind Domain III of serum albumin.

[0500]

[0322] The sdAbs can be from any desired species, such as camelid (e.g, llama, alpaca, dromedary, camel, guanaco), rodent (e.g., mouse, rat), lagomorph (e.g., rabbit), chicken, pig, human, non-human primate, shark, cartilaginous fish, bovine (e.g, cow, bison) and the like. Typically, the sdAb is camelid, CDR-grafted, humanized, or human. For example, as described and exemplified herein, humanized sdAbs can be prepared from camelid VHHs using suitable methods. Exemplary sdAbs derived from a camelid (e.g., a llama) can comprise or consist of any one of SEQ ID NO: 80-149, or an amino acid that has at least about 80% identity to SEQ ID NO: 80-149.

[0501]

[0323] In some instances, the sdAbs are of human origin. Such sdAbs can, for example, can be obtained from human immunoglobulin variable domain (VH or VL) libraries or from mice that express antibodies from human genes.

[0502]

[0324] sdAbs disclosed herein can preferably be humanized. As is well-known in the art, a non-human antibody is typically humanized to reduce the immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, humanized sdAbs comprise one of more CDRs (or portions thereof) that are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. Some FR residues in humanized sdAbs can be substituted with corresponding residues from a non-human antibody (the so-called donor antibody). For instance, non-human CDRs (e.g., CDRs derived from a llama or a camelid) can be grafted into one or more human framework regions (“FR”). The sdAbs disclosed herein can comprise CDR1, CDR2, and CDR3 derived from a non-human sdAb, such as a camelid or any other suitable species, and FW regions derived from a suitable human antibody or the human germline. One or more of the framework regions (i.e., FR1, FR2, FR3, FR4) can further comprise one or more amino acid substitutions or replacements, such as ‘back mutations’ which replace an amino acid residue in the framework region of human origin with a residue from the corresponding position of the donor antibody. One or more mutations in the framework region can be made, including deletions, insertions, and substitutions of one or more amino acids. Variants can 52

[0503] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0504] be produced by a variety of suitable methods. Suitable methods for designing and producing humanized antibodies are well-known in the art, and can readily be adapted to produce humanized sdAbs.

[0505]

[0325] The sdAbs disclosed herein can have half-lives that are comparable to sdAbs that do not compete for binding with FcRn. The sdAbs disclosed herein have the ability to extend the serum half-life, for example when part of a fusion polypeptide. The fusion polypeptide that comprises the sdAbs disclosed herein can have a half-life of at least about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hour, about 3 hours, about 2 hours, or about 1 hour.

[0506]

[0326] The sdAbs disclosed herein can be prepared using any suitable method. A variety of methods have been described and are known in the art. Antibodies and antibody fragments can be prepared by immunization. See, e.g., Kohler et al., (1975), Nature, 256: 495-497; Kohler et al., (1976), Eur. J.

[0507] Immunol., 6:511-519; Milstein et al., (1977), Nature, 266, 550-552; U.S. Patent No., 4,172,124; Harlow et al., (2014), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. For example, any suitable animal (e.g., llama or camel) can be immunized with a suitable antigen (e.g., human serum albumin). Antibody producing cells can be isolated to form a library using conventional methods. The library can be used to screen and identify sdAbs having the desired (e.g., specificity or affinity). sdAbs disclosed herein can be prepared using various phase display methods known in the art. See, e.g., Brinkman et al., (1995), J. Immunol. Methods, 182, 41-50; Ames et al., (1995), J. Immunol. Methods, 184, 177-186; Kettleborough et al. (1994), Eur. J. Immunol., 24, 952-958; Persic et al., (1997), Gene, 187, 9-18; and Burton et al., (1994), Advances in Immunology, 57, 191-280.

[0508]

[0327] As disclosed herein, the sdAbs are preferably humanized. Humanizing the sdAbs disclosed herein can be prepared using any suitable method. The CDRs can be derived from a suitable antibody which binds serum albumin. Sources of suitable CDRs include natural and artificial serum albumin antibodies or binding fragments obtained from human or non-human sources, such as camel, llama, mouse, rat, rabbit, chicken, pig, monkey. The FRs of a humanized antigen binding fragment are preferably of human origin, and can be derived from any human antibody variable region having sequence similarity to the analogous region. Other FR regions of human origin include human variable region consensus sequences. Other FRs can be from other origins such as FR regions encoded by germline antibody gene segments from any suitable species, such as, but not limited to horse, cow, dog, cat, llama, camel.

[0509] 53

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[0511]

[0328] Generally, the sdAbs disclosed herein are not found in naturally occuring species, e.g., humans, camel, llama, rat, monkey.

[0512]

[0329] Binding specificity of the sdAbs disclosed herein can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, EIA, BLI, or BIACORE.

[0513]

[0330] The sdAbs disclosed herein can bind to serum albumin with any desired affinity (i.e., equilibrium dissociation constant (KD)), on rate and off rate. The affinity, on rate (Kon or ka) and off rate (Koff or kd) can be selected to obtain a maximal serum half-life. Generally, a fast on rate and a fast or moderate off rate for binding to serum albumin is preferred. sdAbs disclosed herein with these characteristics will quickly bind serum albumin after being administered, and upon dissociation will rebind serum albumin rapidly. Additionally, the affinity, on rate (Kon or ka) and off rate (Koff or kd) can be selected to obtain a moderate serum half-life (slower on rate and / or faster off rate).

[0514]

[0331] The antigen binding fragment disclosed herein generally bind serum albumin with a KD of about 0.1 nM to about 50 µM. In some embodiments, the antigen binding fragment binds serum, albumin with a KD (KD = Koff(kd) / Kon(ka)) of about 0.1 to 1 nM, about 0.1 to 5 nM, or about 10 to 50 nM, as determined by Bio-Layer Interferometry (BLI) or surface plasmon resonance (e.g., using a BIACORE instrument). In embodiments, the fusion proteins disclosed herein.

[0515] D. Non-Immunoglobulin Scaffolds

[0516]

[0332] This disclosure also relates to compounds that bind human serum albumin that comprise a non-immunoglobulin scaffold and CDRs of any of the sdAbs described herein. Suitable non-immunoglobulin scaffolds and methods for grafting CDRs onto the scaffolds are well-known. For example, suitable scaffolds include the Z domain of protein A, ankyrin repeats, Kunitz domains, lipocalins and the like. See, e.g., Frejd et al (2017), Exp Mol Med., 49(3):e306.; Pluckthun et al., (2015), Ann. Rev. of Pharmacology and Toxicology, 55:489-511. In exemplary embodiments, this disclosure relates to a binding domain that binds human serum albumin and comprises a non-immunoglobulin scaffold and any of the CDRs (CDR1, CDR2 and CDR3) shown in Table 1, for example, SEQ ID NO:7, SEQ ID NO:38 and SEQ ID NO:22; or SEQ ID NO:37, SEQ ID NO:9 and SEQ ID NO:12; or SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:12; or SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12; or SEQ ID NO:8, SEQ ID NO:11 and SEQ ID NO:12; or SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; or SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

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[0519] E. Polypeptides and Compositions Comprising sdAbs

[0520]

[0333] The sdAbs and non-immunoglobulin fragments disclosed herein can be formatted into a variety of suitable polypeptide structures, for example, proteins, fusion proteins, conjugates or any other suitable format.

[0521] i. Fusion Proteins And Conjugates

[0522]

[0334] The sdAbs and non-immunoglobulin scaffolds disclosed herein are particularly suitable for use in preparing fusion polypeptides and conjugates, e.g., that have extended serum half-life. The fusion polypeptide can comprise a continious polypeptide chain that comprises a sdAb or non-immunoglobulin format as disclosed herein and an amino acid sequence of interest, typically a therapeutic protein or peptide. The sdAb can be fused to the amino acid sequence of interest directly, or indirectly, for example through an amino acid linker. If desired, the fusion polypeptide can associate with one or more other polypeptides to form a functional polypeptide complex. For example, when it is desired to provide an extended half-life form of a protein that is a heterodimer, a fusion polypeptide that contains a sdAb disclosed herein and one member of the heterodimer can be prepared, and it can associate with the other member of the heterodimer to form a functional polypeptide complex.

[0523]

[0335] In non-limiting examples, the fusion polypeptide can be represented by Formula V or VI:

[0524] [A1]-[L1]-[D1] (V)

[0525] [D1]-[L1]-[A1] (VI),

[0526] wherein A1 is the sdAb disclosed herein, L1 is a linker that connects or links A1 to D1 or is absent, and D1 is an amino acid sequence of interest.

[0527]

[0336] The amino acid sequence of interest can be any desired amino acid sequence, typically it is the amino acid sequence of a therapeutic agent, diagnostic agents, or targeting agent. Exemplary amino acid sequences of interest include, but are not limited to those of, cytokines, with the proviso that the cytokine is not IL-2 and / or IL-21, receptors (e.g., soluble receptors that bind cytokines, growth factors, hormones and the like), enzymes, growth factors, hormones (e.g., peptide hormones), chemokine, tumor antigen binding domains, matrix proteins, pathogen antigens, immunoglobulin formats (e.g., antigen-binding portions of antibodies) or fragments thereof, antibiotics, cytotoxic drugs, or other recombinant polypeptide complexes.

[0528]

[0337] Suitable amino acid sequences of interest (e.g., cytokines, growth factors, hormones, and other polypeptides) include, but are not limited to, 4-1BBL, ApoE, Apo-SAA, BDNF, cardiotrophin-1, CD40L, CD70, EGF, EGF receptor, ENA-79, exotoxin, Epo-R, GF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16,

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[0531] FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, fibroblast growth factor 10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GITRL, GM-CSF, CF-beta1, insulin, IFN-alpha, IFN-beta, IFN-gamma, IGF-I, IGF-II, IL-1, IL-4, IL-5, IL-6, IL-10, IL-18, IL-22, IL-23p19, IL-11, IL-12, IL-13, IL-15, IL-12p35, IL-30 (IL27p28), IL-34, IL-35, IL-36, inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-I (MCAF), MCP-2, MCP-3, MCP- 4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-Ia, MIP-I β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor- 1 (MPIF-I), NAP -2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, OX40L, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFl α, SDFl β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNIL-I, TPO, VEGF, GCP-2, GRO / MGSA, GRO-β, GRO-γ, HCCl, 1-309, TNF-alpha, IFN-gamma, MMP- 12, CEA, MMP- 12, PsmAr,. Preferably, the cytokine is not IL-2 and / or IL-21.

[0532] Preferably, the cytokine is not IL-2. Preferably, the cytokine is not IL-12. It will be appreciated that this list is by no means exhaustive.

[0533]

[0338] Suitable hormones and hormone receptor agonists, include, Leptin, GLP-1, GLP-1 receptor agonists, GIP, Ghrelin, asprosin, CGRP, insulin and the like.

[0534]

[0339] Suitable chemokines can include, for example, CXCL9, CXCL10, CXCL111, CXCL13, CCL19, CCL20, CCL21, XCL1 or functional fragments.

[0535]

[0340] Suitable tumor antigen binding domains can include, but are not limited to domains that bind EpCAM, EGFR, HER-2, HER-3, c-Met, FolR, PSMA, CD38, BCMA, CEA, 5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, WT1.

[0536]

[0341] Examples of suitable receptors include, but are not limited to, Type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type I IL receptors, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; Type II Cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), Type II IL receptors; chemokine receptors, such as CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, XC chemokine receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors,

[0537] 56

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[0539] such as TGF-beta receptor 1, TGF-beta receptor 2; Ig super family receptors, such as IL-1 receptors, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, internalizing receptors that are over-expressed on certain cells, such as the epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor cells, an internalizing cellular receptor, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF receptor, human chemokine receptors CXCR4 or CCR5, and HER 1, HER 2, HER 3, HER 4, and CD4.

[0540]

[0342] Examples of pathogenic antigens include, but are not limited to, an antigen of Helicobacter pylori, an antigen of Mycobacterium tuberculosis, and an antigen of influenza virus, and non-structural protein type 3 (NS3) from the hepatitis C virus. Examples of matrix proteins include, but are not limited to, an extracellular matrix protein, elastin, fibronectin, and laminin.

[0541]

[0343] The amino acid sequence of interest preferably encodes a cytokine, with the proviso that the cytokine is IL-2 and / or IL-21. Suitable cytokines, include for example, IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-4, IL-5, IL-6, IL-10, IL-18, IL-22, IL-23p19, IL-11, IL-12, IL-13, IL-15, IL-12p35, IL-30 (IL27p28), IL-34, IL-35, IL-36 and the like. Preferable cytokines include, IL-12 (IL-12 p35 and / or IL-12p40), IL-10, and IL-18. Preferably, the cytokine is not IL-2 and / or IL-21. Preferably, the cytokine is not IL-2. Preferably, the cytokine is not IL-12.

[0542]

[0344] The amino acid sequence of interest can be an immunoglobulin format, such as another sdAb that binds to any desired target. Suitable immunoglobulin formats are well-known in the art and include bispecific and multi-specific formats (i.e., bind HSA and at least one other target) that can be bivalent or multivalent. Suitable immunoglobulin formats include, for example, heavy chain antibody, Fab, Fab’, F(ab’)2, Fv, disulfide stabilized Fv fragment (dsFv), (dsFV) 2, bispecific dsFv (dsFv-dsFv’), a disulfide diabody, a single-chain Fv (scFv), a diabody, triabodies, tetrabodies, tribodies, a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, sdAb, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete IgG antibody structure (e.g., an antibody fragment from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody).

[0543]

[0345] The amino acid of interest can be a immunoglobulin formats that are known in the art as bispecific, trispecific or multispecific cell engagers (or an amino acid chain of a such a cell engager). Such cell engagers, including bispecific T-cell engagers (BiTEs), dual-affinity re-targering molecules (DARTs), bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs) are well-known in the art and include a binding region (typically a sdAb or two variable regions that form a VH / VL binding sight) that binds an antigen on an immune cell (e.g, an effector cell such as a T-cell or 57

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[0545] NK cell) and a binding region that binds a tumor-associated antigen. See, e.g., Allen et al., (2021), Life (Basel), 11(6):465. For example, such bispecific, trispecific or multispecific cell engagers can engage T-cells and tumor cells, by binding to CD3 on the T cell and to a tumor associated antigen on a tumor cell. For example, such bispecific, trispecific or multispecific cell engagers can engage NK cells and tumor cells, by binding to CD16 on the NK cell and to a tumor associated antigen on a tumor cell.

[0546]

[0346] Of specific interest are fusion polypeptides that are suitable for use with payloads that target or are targeted to tumor microenvironments.

[0547]

[0347] This disclosure also relates to conjugates. Like the fusion polypeptides described herein, conjugates comprise a sdAb as described herein and an amino acid sequence of interest. In the conjugates of this disclosure, the sdAb is covalently bonded directly or indirectly to the amino acid sequence of interest. The sdAb can be conjugated to the amino acid sequence of interest at the C-terminus of the amino acid sequence of interest, at the N-terminus of the amino acid sequence of interest, or internally, for example through, an amino acid side chain of an amino acid in the amino acid sequence of interest. Many suitable linkers and functional groups that can be used to prepare conjugates as described herein are well-known in the art. For example, homo- or hetero-bifunctional cross-linkers can be used to link a sdAb disclosed herein to an amino acid sequence of interest, for example through an amine group on the sdAb and a carboxyl group on the amino acid sequence of interest. In some aspects, the sdAb is indirectly conjugated to the amino acid sequence of interest through a linker that includes a peptide or polypeptide amino acid sequence. Such linkers can be non-cleavable or cleavable, for example by protease (e.g., a protease with activity that is associated with diseased cells or tissues as disclosed herein).

[0548]

[0348] The sdAb and the amino acid sequence of interest in the fusion polypeptides and conjugates of this disclosure can be linked to each other directly or indirectly. The sdAb and the amino acid sequence of interest may be linked through a suitable amino acid or peptide or polypeptide linker. Such linker sequences can be a naturally occuring sequence or a non-naturally occuring sequence. Preferably, the linker sequence is a non-naturally occuring sequence.

[0549]

[0349] The sdAb and the amino acid sequence of interest can be linked by a non-cleavable linker. The sdAb and the amino acid sequence of interest is preferably linked by a cleavable linker. The cleavable linker can comprise one or more cleavage sites for one or more desired protease. Preferably, the desired protease activity is enriched or selectively expressed at the desired target site of the amino acid sequence of interest, such as a desired cite of cytokine activity (e.g., the tumor microenvironment). The linker is preferentially or selectively cleaved at the target site.

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[0552]

[0350] Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 83, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99m or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.

[0553]

[0351] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked amino acid sequence of interest. In a preferred embodiment, the linker is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the linker comprises a protease cleavage site. In some cases, the linker comprises one or more cleavage sites. The linker can comprise a single protease cleavage site. The linker can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases the linker comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases. A linker comprising two or more cleavage sites is referred to as a “tandem linker.” The two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.

[0554]

[0352] Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation.

[0555]

[0353] Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases,

[0556] 59

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[0558] metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPα), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14). In addition, or alternatively, the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and / or Cathepsin L. Preferably, the linker can be cleaved by MMP14 or Cathepsin L.

[0559]

[0354] Proteases useful for cleavage of linkers and for use in the fusion polypeptides and conjugates disclosed herein, including half-life extended cytokines disclosed herein are presented in Table 3, and exemplary proteases and their cleavage site are presented in Table 4.

[0560] Table 3. Proteases relevant to inflammation and cancer

[0561] Protease Specificity Other aspects

[0562] Secreted b killer T cells:

[0563] nt

[0564] l

[0565]

[0566] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0567] Protease Specificity Other aspects Mucosa-associated Cleaves after arginine Type of cysteine protease; likely acts both lmhoid tissue residues as a scaffold and roteolticall active

[0568] e;

[0569] ;

[0570] e

[0571] e

[0572] n

[0573] n

[0574]

[0575] 61

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[0577] Protease Specificity Other aspects process of numerous inflammation- associated diseases

[0578]

[0579] Protease Cleavage Domain Sequence SEQ ID NO:

[0580] MMP7 KRALGLPG

[0581]

[0582] 62

[0583] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0584] Protease Cleavage Domain Sequence SEQ ID NO:

[0585] HIV Protease GVSQNYPIVG266

[0586] [3

[0587]

[0588] 55] Exemplary protease cleavable linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease. Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPα cleavable linkers, and cathepsin cleavable linkers. Some preferred protease-cleavable linkers are cleaved by a MMP and / or a cathepsin.

[0589]

[0356] Exemplary linkers that may be suitable for the fusion polypeptide disclosed herein are disclosed in International Publication No.: WO2020 / 232305. For example, the linker can comprise the sequence 63

[0590] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0591] GPAGLYAQ (SEQ ID NO: 214); GPAGMKGL (SEQ ID NO: 215); PGGPAGIG (SEQ ID NO: 216); ALFKSSFP (SEQ ID NO: 217); ALFFSSPP (SEQ ID NO: 218); LAQRLRSS (SEQ ID NO: 219);

[0592] LAQKLKSS (SEQ ID NO: 220); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 221); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 222);

[0593] KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 223);

[0594] RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 224);

[0595] KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 225);

[0596] SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 226);

[0597] RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 227);

[0598] GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 228);

[0599] RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 229);

[0600] RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 230);

[0601] RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 231);

[0602] GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 232);

[0603] GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 233);

[0604] RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 234);

[0605] RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 235);

[0606] SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 236);

[0607] KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 237);

[0608] KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 238); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 239)

[0609]

[0357] Certain preferred fusion polypeptides comprise the sequence GPAGLYAQ (SEQ ID NO: 214) or ALFKSSFP (SEQ ID NO: 217).

[0610]

[0358] Preferred linkers comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NOs: 214 to 239. In some embodiments, the linker comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 214 to 239.

[0611]

[0359] The linker can comprise one or more cleavage motif or functional variants that are the same or different. The linker can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants. Linker comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more. A “functional variant” of a linker retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional 64

[0612] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0613] variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising any one of SEQ ID NOs: 214 to 239.

[0614]

[0360] The length, degree of flexibility and other properties of the linkers used in the fusion polypeptide may have some influence on properties, including, but not limited to the affinity, the specificity, or avidity for one or more components. In some instances, the linker can comprise flexible residues (such as glycine or serine) so that the adjacent sdAbs or amino acid sequence of interest are free to move relative to each other.

[0615]

[0361] Other linker considerations include, but are not limited to, the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability, rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome.

[0616]

[0362] In some instances, a non-peptide linker may be suitable. For example, such linkage can be through covalent binding, affinity binding, intercalation, coordinate binding, and complexation. Covalent linking can be achieved by direct condensation of existing side chains or by the incorporation of external bridging molecules. Linking agents can be useful for linking the sdAb thereof and the amino acid sequence together. For example, representative coupling agents include organic compounds such as thioesters, carbodiimide, succinimide esters, diisocyanate, glutaraldehyde, diazobenzenes, and hexamethylene diamines. Non-peptide linkers are well established in the art and may be suitable for the fusion polypeptide described herein.

[0617]

[0363] The linker desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0618]

[0364] In some embodiments, the linker is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location. The linker is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the linker is cleaved resulting in separation of the linked domain.

[0619]

[0365] The fusion polypeptide can comprise a sdAb disclosed herein and an amino acid sequence of interest. For example, the fusion polypeptide can comprise a sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of the foregoing; a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 5, 9, 10, 11, 38 or a variant of any of the foregoing; and a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of 65

[0620] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0621] the foregoing; and an amino acid of interest. Preferably, the amino acid sequence of interest encodes a cytokine (e.g., IL-12, IL-10, or IL-18). Preferably, the cytokine is not IL-2 and / or IL-12 and / or IL-21. Preferably, the cytokine is not IL-2. Preferably, the cytokine is not IL-12. The sdAb and the amino acid of interest, for example, encoding a cytokine are operably linked. Preferably, the sdAb and the amino acid of sequence of interest, for example, encoding a cytokine, are preferably linked by a cleavable linker. The cleavable linker is preferably a protease cleavable linker.

[0622]

[0366] The fusion polypeptide can comprise a sdAb comprising any of the sdAbs disclosed herein and an amino acid sequence of interest. For example, the fusion polypeptide can comprise a sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of the foregoing; a SEQ ID NO: 2, 5, 9, 10, 11, 38 or a variant of any of the foregoing; a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of the foregoing; a FR1 comprising the amino acid sequence of any of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO:15-19, 24-28, 32, 33, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof; and an amino acid of interest. Preferably, the amino acid sequence of interest encodes a cytokine, (e.g., IL-12, IL-10, or IL-18), with the proviso that the cytokine is not IL-2 and / or not IL-21. Preferably, the cytokine is not IL-2 and / or IL-21. Preferably, the cytokine is not IL-2. Preferably, the cytokine is not IL-12. The sdAb and the amino acid of interest, for example, encoding a cytokine are operably linked. Preferably, the sdAb and the amino acid of sequence of interest, for example, encoding a cytokine, are preferably linked by a cleavable linker. The cleavable linker is preferably a protease cleavable linker.

[0623]

[0367] The fusion polypeptide can comprise a sdAb comprising a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or 66

[0624] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0625] consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or

[0626] 67

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[0628] consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0629]

[0368] The fusion polypeptide can comprise a sdAb comprising the amino acid sequence of any one of SEQ ID NO: 39-58, 60-79, or 286-290, and an amino acid sequence interest, such an amino acid sequence encoding a cytokine, with the proviso that the cytokine is not IL-2 and / or IL-21.

[0630]

[0369] The fusion polypeptide can comprise a sdAb comprising or consisting of the amino acid sequence of a) SEQ ID NO: 39 or variant thereof; b) SEQ ID NO: 40 or a variant thereof; c) SEQ ID NO: 41, or a variant thereof; d) SEQ ID NO: 42 or a variant thereof; e) SEQ ID NO: 43, or a variant thereof; f) SEQ ID NO: 44 or a variant thereof; g) SEQ ID NO: 45 or a variant thereof; h) SEQ ID NO: 46 or a variant thereof; i) SEQ ID NO: 47 or variant thereof; j) SEQ ID NO: 48, or a variant thereof; k) SEQ ID NO: 49 or a variant thereof; l) SEQ ID NO: 50 or a variant thereof; m) SEQ ID NO: 51 or a variant thereof; n) SEQ ID NO: 52 or a variant thereof; o) SEQ ID NO: 53 or a variant thereof; o) SEQ ID NO: 54 or a variant thereof; p) SEQ ID NO: 55 or a variant thereof; q) SEQ ID NO: 56 or a variant thereof; r) SEQ ID NO: 57 or a variant thereof; s) SEQ ID NO: 58 or a variant thereof; t) SEQ ID NO: 60 or a variant thereof; u) SEQ ID NO: 61 or a variant thereof; v) SEQ ID NO: 62 or a variant thereof; w) SEQ ID NO: 63 or a variant thereof, x) SEQ ID NO: 64 or a variant thereof, y) SEQ ID NO: 65 or a variant thereof; z) SEQ ID NO: 66 or a variant thereof; za) SEQ ID NO: 67 or a variant thereof; zb) SEQ ID NO: 68 or a variant thereof; zc) SEQ ID NO: 69 or a variant thereof; zd) SEQ ID NO: 70 or a variant thereof; ze) SEQ ID NO: 71 or a variant thereof; zf) SEQ ID NO: 72 or a variant thereof; zg) SEQ ID NO: 73 or a variant thereof; zh) SEQ ID NO: 74 or a variant thereof; zi) SEQ ID NO: 75 or a variant thereof; zj) SEQ ID NO: 76 or a variant thereof; zk) SEQ ID NO: 77 or a variant thereof; zl) SEQ ID NO: 78 or a variant thereof; or zm) SEQ ID NO: 79 or a variant thereof. Preferably, the fusion polypeptide does not comprise a sdAb having the amino acid sequence of SEQ IN NO: 59.

[0631]

[0370] Additional moieties and / or linkers can be present, as appropriate. Additional specific applications of uses for the sdAbs are disclosed herein.

[0632] ii. Half-Life Extended Cytokines

[0633]

[0371] Exemplary fusion polypeptides and conjugates disclosed herein are half-life extended cytokines. The half-life extended cytokines can be a fusion polypeptide or a conjugate that comprises a sdAb or non-immunoglobulin format as disclosed herein and a cytokine polypeptide, and optionally a linker (e.g., cleavable or non-cleavable linker).

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[0636]

[0372] Any cytokine of interest can be suitable for inclusion in a half-life extended cytokine of this disclosure. Exemplary cytokines include, but are not limited to, interleukins such as IL-7, IL-10, IL-12, IL-15, IL-18, IL-23, IL-36, IFN-alpha (e.g., human IFN-alpha1, human IFN-alpha2, human IFN-alpha4, human IFN-alpha5, human IFN-alpha6, human IFN-alpha7, human IFN-alpha8, human IFN-alpha10, human IFN-alpha13, human IFN-alpha14, human IFN-alpha16, human IFN-alpha17, human IFN-alpha2), IFN-beta, IFN-kappa, or IFN-epsilon, lymphotoxin, TGF-beta1, TGFbeta2, TGFbeta3, GM-CSF, CXCL10, CCL19, CCL20, CCL21, TNF, 4-1BBL, CD40L, OX40L, CD70 and functional fragments or muteins of any of the foregoing. Preferred cytokines for use in the half-life extended cytokines disclosed herein are IL-12, muteins, functional variants, and functional fragments, or subunits of any of the foregoing.

[0637]

[0373] The half-life extended cytokines can further comprise a linker as described herein. The linker can be a cleavable linker as disclosed herein. Preferably, the cleavable linker is a protease cleavable linker. The desired protease activity is enriched or selectively present at the desired target site of the cytokine activity (e.g., the tumor microenvironment). The linker is preferentially or selectively cleaved at the target site.

[0638] ii. Half-Life Extended Immunoglobulin Formats

[0639]

[0374] Exemplary fusion polypeptides and conjugates disclosed herein are half-life extended immunoglobulin formats. The half-life extended immunoglobulin format can be a fusion polypeptide or a conjugate that comprises a sdAb or non-immunoglobulin format as disclosed herein and an immunoglobulin format, and optionally a linker (e.g., cleavable or non-cleavable linker). Suitable immunoglobulin formats are well-known in the art and examples of suitable immunoglobulin formats are disclosed herein. In certain examples, the half-life extended immunoglobulin format includes a bispecific, trispecific or multispecific cell engager, such as a BiTE, DART, BiKE or TriKE. For example, the half-life extended immunoglobulin format can comprise a sdAb or non-immunoglobulin format that binds HSA as described herein, and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on a T cell (e.g., CD3) and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on a target cell, such as a tumor associate antigen. In other examples, the half-life extended immunoglobulin fragment can comprise a sdAb or non-immunoglobulin format that binds HSA as described herein, and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on a NK cell (e.g., CD16) and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on a target cell, such as a tumor associate antigen. As is well-known in the art, such bispecific, trispecific or multispecific cell engagers can comprise more than one polypeptide chain.

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[0642] The sdAbs or non-immunoglobulin formats disclosed herein can be linked directly or indirectly to the immune cell (e.g., T cell or NK cell) binding domain and / or the target antigen (e.g., tumor associated antigen) binding domain. The sdAbs disclosed herein can be linked to the immune cell (e.g., T cell or NK cell) binding domain and / or the target antigen (e.g., tumor associated antigen) binding domain through a linker, such as a protease cleavable linker as described herein. The sdAbs disclosed herein can be linked to the immune cell binding domain. The sdAbs disclosed herein can be linked to the target antigen binding domain. The sdAbs disclosed herein can be linked to both the immune cell binding domain and the target antigen binding domain. The half-life extended bispecific, trispecific or multispecific cancel engager can comprise a) a sdAb or non-immunoglobulin format as disclosed herein that binds HSA, b) a binding region (e.g., sdAb, scFv etc.) that has specificity for the TCR complex including CD3, and c) a binding region that has specificity for a target antigen, such as a tumor associated antigen.

[0643]

[0375] The binding region with specificity for the TCR complex can specifically bind to CD3, preferably human CD3. The binding region can have binding specificity for one or more chain of the CD3 complex, such as CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. The binding region can have binding specificity for CD3 epsilon. The binding region can have binding specificity for CD3 gamma. The binding region can have binding specificity for CD3 delta. The binding region can have binding specificity for CD3 zeta. The binding region having binding specificity for the TCR can further comprise a domain that specifically bind the alpha chain of the TCR and / or the beta chain of the TCR.

[0644]

[0376] The binding region having binding specificity to CD3 can be any suitable immunoglobulin format that binds CD3. Examples include, but are not limited to, an antigen binding fragments, such as single domain antibodies, Fab, Fab’, F(ab)2, single chain Fv fragments, disulfide stabilizd Fv fragments, Fv fragments, heavy chain monomers or dimers, light chain monomers or dimers. Suitable binding fragments that have specificity for CD3 can be obtained, for example, from any anti-CD3 antibodies. Many such antibodies and binding regions are well-known, such as muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5M, F111-409, CLB-T3.4.2M TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-141, XIII-87, T3 / RW2-8C8, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0645]

[0377] In addition, the half-life extended bispecific, trispecific or multispecific cell engagerfurther comprises a a region (e.g., sdAb, scFv) that binds to a target antigen. A target antigen is involved in and / or associated with a disease, disorder, or condition. In particular, the target antigen can be associated with or involved with a disease, disorder, or condition. Specifically, the target antigen can be associated with cancer, an inflammatory disease, an immunological disorder, an autoimmune disease, a viral disease,

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[0648] or a bacterial disease. The target antigen binding region can be any suitable immunoglobulin format that binds to the target antigen. Examples include, but are not limited to, a antigen binding fragments, such as single domain antibodies, Fab, Fab’, F(ab)2, single chain Fv fragments, disulfide stabilizd Fv fragments, pv fragments, heavy chain monomers or dimers, light chain monomers or dimers.

[0649] F. Inducible Cytokine Prodrugs

[0650]

[0378] This disclosure relates to inducible cytokine prodrugs. The inducible cytokine prodrugs include a sdAb or non-immunoglobulin format that binds HSA, to extend the half-life of the inducible cytokine prodrug as described herein, a cytokine polypeptide and a protease cleavable linker. Typically, the inducible cytokine prodrug further comprises a moiety or domain that binds the cytokine polypeptide (i.e., a blocking domain) and attenuates cytokine activity. The sdAb can also contribute to attenuation, for example through steric effects. The blocking domain is capable of blocking all or some of the receptor agonist activity of the cytokine by noncovalently binding to the cytokine (e.g., to IL-2, IL-12, or IFN) and / or sterically blocking receptor binding, for example. Upon cleavage of the protease cleavable linker a form of the cytokine is released that is active (e.g., more active than the inducible cytokine prodrug).

[0651]

[0379] The prodrug includes protease cleavage sequences, which are cleaved by proteases that are associated with, and are typically enriched or selectively present in, the tumor microenvironment.

[0652]

[0380] Typically, the released cytokine is at least 10 x more active than the cytokine polypeptide prodrug. Preferably, the released cytokine is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible cytokine prodrug.

[0653]

[0381] The form of cytokine that is released upon cleavage of the inducible cytokine prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring cytokine. Even though the half-life of the inducible cytokine prodrug is extended, toxicity is reduced or eliminated because the agonist activity of the circulating inducible cytokine prodrug is attenuated and active cytokine is targeted to the desired site of activity (e.g., tumor microenvironment). The inducible cytokine prodrugs of this disclosure overcome the toxicity and short half-life problems that have severely limited the clinical use pf cytokines in oncology.

[0654]

[0001] Any cytokine of interest can be suitable for the inducible cytokine polypeptide prodrug of this disclosure with the proviso that the cytokine polypeptide is not IL-21. Exemplary cytokines include, but are not limited to, interleukins such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-23, IL-36, IFN-alpha (e.g., human IFN-alpha1, human IFN-alpha2, human IFN-alpha4, human IFN-alpha5, human IFN-alpha6,

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[0657] human IFN-alpha7, human IFN-alpha8, human IFN-alpha10, human IFN-alpha13, human IFN-alpha14, human IFN-alpha16, human IFN-alpha17, human IFN-alpha2), IFN-beta, IFN-kappa, or IFN-epsilon, lymphotoxin, TGF-beta1, TGFbeta2, TGFbeta3, GM-CSF, CXCL10, CCL19, CCL20, CCL21 and functional fragments or muteins of any of the foregoing. Preferred cytokines for use in the inducible cytokine prodrugs disclosed herein are IL-2, IL-12, IFN, muteins, functional variants, and functional fragments, or subunits of any of the foregoing. Preferably, the cytokine polypeptide is not IL-21.

[0658]

[0382] Inducible cytokine prodrugs have been described, for example, in International Publication Nos.: WO2019 / 222294, WO2019 / 222295, WO2019 / 222296, WO2021 / 097376.

[0659]

[0383] It will be appreciated by those skilled in the art, that the number of polypeptide chains, and the location of the elements, the sdAb, the protease cleavable linker(s), and the blocking domain, when present, (and components of such elements, such as a VH or VL domain) on the polypeptide chains can vary and is often a matter of design preference. All such variations are encompassed by this disclosure.

[0660]

[0384] The inducible cytokine prodrug can comprise a single polypeptide chain. Typically, the single polypeptide chain comprises a cytokine polypeptide [A], a sdAb or non-immunoglobulin format that binds HSA as described herein [H], typically a blocking domain [D], and a protease cleavable linker [L]. When the blocking domain is absent it is preferred that the sdAb or non-immunoglobulin format also functions as a blocking domain as described herein and sterically inhibits binding of the cytokine polypeptide to the cytokine receptor. The cytokine polypeptide [A] can be operably linked to the sdAb or non-immunoglobulin format, the blocking domain (when present), or both the sdAb and blocking domain (when present) by a protease cleavable linker. Typically, the single polypeptide chain comprises one cytokine polypeptide or two cytokine polypeptides. The cytokine polypeptide can be located at any desired position in the single polypeptide chains.

[0661]

[0385] The single polypeptide can comprise two or more sdAbs that also function as a blocking domain. When two or more of such sdAbs are present in the inducible cytokine prodrug, they can block all or some of the receptor agonist activity of the cytokine and also extend serum half-life. When two or more sdAbs are present in the inducible cytokine prodrug, a separate blocking domain is optional and is typically not present.

[0662]

[0386] The inducible cytokine prodrug can be of any of Formulas (I)-(VI):

[0663] [A]-[L1]-[H]-[L2]-[D] (I);

[0664] [D]-[L2]-[H]-[L1]-[A] (II);

[0665] [A]-[L1]-[D]-[L2]-[H] (III);

[0666] [H]-[L2]-[D]-[L1]-[A] (IV);

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[0669] [H]-[L1]-[A]-[L2’]-[D] (V);

[0670] [D]-[L1]-[A]-[L2’]-[H] (VI);

[0671] [H]-[L1]-[A]-[L2]-[H] (VII).

[0672]

[0387] In Formulas (I) – (VII), [A] is a cytokine polypeptide, [D] is a blocking domain, [H] is a sdAb or non-immunoglobulin format that binds HSA as disclosed herein, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. [L1] and [L2] or [L1] and [L2’] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired. In some instances, the blocking domain can comprise a sdAb or a non-immunoglobulin format that binds to HSA. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 214) or ALFKSSFP (SEQ ID NO: 217), particularly ALFKSSFP (SEQ ID NO: 217). Preferably, each of [L1] and [L2’] comprises SEQ ID NO: 214 or 217.

[0673]

[0388] The inducible cytokine prodrugs can contain two or more polypeptide chains. Such inducible cytokine prodrugs comprise a cytokine polypeptide, a sdAb that extends the half-life of the inducible cytokine prodrug, typically a blocking domain, and a protease cleavable linker. The components of the inducible cytokine prodrug can be on the same polypeptide chain or on different polypeptide chains. Illustrative of this, and as disclosed and exemplified herein, components of the blocking domain can be present on separate polypeptide chains. For example, a first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form an antigen-binding site, such as a Fab that binds to the cytokine (e.g., IL-2, IL-12, or IFN) and attenuates the cytokine activity.

[0674]

[0389] For example, the inducible cytokine prodrug can have a) a first polypeptide of Formulas (I)-(VII), wherein [A] is a cytokine polypeptide, [D] is an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH), [H] is a sdAb or non-immunoglobulin format that binds HSA as disclosed herein, [L1] is a protease-cleavable polypeptide linker, [L2] is an polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker; and b) a second polypeptide chain comprising an antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH. [L1] and [L2] or [L1] and [L2’] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired.

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[0677]

[0390] For example, the inducible cytokine prodrug can have a) a first polypeptide of Formulas (I)-(VI), wherein [A] is a cytokine polypeptide, [D] is an antibody light chain (VL + CL) or light chain variable domain (VL), [H] is a sdAb or non-immunoglobulin format that binds HSA as disclosed herein, [L1] is a protease-cleavable polypeptide linker, [L2] is an polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker; and b) a second polypeptide an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is complementary to the VL + CL or VL. [L1] and [L2] or [L1] and [L2’] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired.

[0678]

[0391] For instances, the first polypeptide chain can comprise from the amino terminal to the carboxy terminal the cytokine polypeptide, a protease cleavable linker, a sdAb or non-immunoglobulin format that binds HSA as disclosed herein, and a VH and CH1 of an antibody that binds the cytokine (e.g., IL-2, an IL-12 subunit i.e., p35, p40, or the p35p40 heterodimeric complex, or IFN). The second polypeptide chain can comprise a VL and CL of an antibody that binds the cytokine (e.g., IL-2, IL-12 subunit (i.e., p35, p40, or the p35p40 heterodimeric complex), or IFN) and that together with the VH and CH1 of the first polypeptide chain form a Fab that binds the cytokine (e.g., IL-2, IL-12 subunit (i.e., p35, p40, or the p35p40 heterodimeric complex), or IFN) polypeptide.

[0679]

[0392] Inducible cytokine prodrugs of cytokines that comprise two subunits, such as IL-12, can also comprise two or more different polypeptides. For instance, the first polypeptide can comprise a first cytokine subunit (e.g., IL-12p35 or IL-12p40), and optionally a blocking domain. The blocking domain, when present, can be operably linked to the first cytokine subunit through a first protease cleavable linker. The second polypeptide chain can comprise a second cytokine subunit operably linked to a sdAb that binds HSA as disclosed herein through a second protease cleavable linker, and optionally a blocking domain. The blocking domain when present can be operably linked to the second cytokine subunit through a protease cleavable linker or can be operably linked to the sdAb that binds HSA through a linker that is optionally protease cleavable. In such embodiments, preferably, only one of the first and second polypeptide contains the blocking domain. Typically, the first polypeptide and second polypeptides contain different cytokine subunits. For instance, when the cytokine is IL-12 and the IL-12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and when the IL-12 subunit in the first polypeptide is p40, the IL-12 subunit in the second polypeptide is p35. A blocking domain can be a single chain antibody that binds the cytokine or an antigen binding fragment thereof. The cleavable linkers in this inducible cytokine prodrug can be the same or different.

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[0682]

[0393] The inducible cytokine polypeptide prodrug can comprise three different polypeptides. One polypeptide chain can comprise a cytokine subunit and a second polypeptide can comprise the other cytokine subunit, and the third polypeptide comprises at least a portion (component) of the blocking domain. The first polypeptide can comprise a cytokine subunit, and optionally a sdAb. The sdAb, when present, can be operably linked to the cytokine subunit through a protease cleavable linker. The second polypeptide can comprise a cytokine subunit, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and optionally a sdAb as disclosed herein. When the sdAb is present, it can be operably linked to the cytokine subunit through a protease cleavable linker and the antibody heavy chain or light chain is either a) operably linked to the IL-12 subunit through a second protease cleavable linker, or b) operably linked to the sdAb through an optionally cleavable linker. The third polypeptide can comprise an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide, or an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms a cytokine binding site. In this complex, the cytokine blocking domain is preferably an antigen binding fragment of an antibody. The antigen binding fragment comprises as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The protease cleavable linkers in this inducible cytokine prodrug can be the same or different.

[0683]

[0394] For example, one polypeptide chain comprises either the p35 or p40 IL-12 subunit, but not both, and a second polypeptide comprises the other IL-12 subunit and the third polypeptide comprises at least a portion (component) of the blocking domain. The first polypeptide can comprise an IL-12 subunit, and optionally a sdAb. The sdAb, when present, can be operably linked to the IL-12 subunit through a protease cleavable linker. The second polypeptide can comprise an IL-12 subunit, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and optionally a sdAb as disclosed herein. When the sdAb is present, it can be operably linked to the IL-12 subunit through a protease cleavable linker and the antibody heavy chain or light chain is either a) operably linked to the IL-12 subunit through a second protease cleavable linker, or b) operably linked to the sdAb through an optionally cleavable linker. The third polypeptide can comprise can an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide, or an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms an IL-12 binding site. When the IL-12 subunit in the first polypeptide is p35, the IL-12 subunit in the second polypeptide is p40, and when the IL-12 subunit in the first polypeptide is p40, the IL-12 subunit in the second polypeptide is p35. In this inducible cytokine 75

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[0685] prodrug, the IL-12 blocking domain is preferably an antigen binding fragment of an antibody. The antigen binding fragment comprises as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The protease cleavable linkers in this inducible IL-12 prodrug can be the same or different.

[0686]

[0395] The inducible polypeptide complex can comprise two different polypeptides wherein cytokine subunits (e.g., p35 and p40) are located on the same polypeptide chain. For example, a first polypeptide chain can comprise p35, p40, a sdAb and at least an antigen binding portion of an antibody light chain. p35 and p40 can be operably linked, and the sdAb can be operably linked to p40 through a first protease cleavable linker and the antigen binding portion of an antibody light chain can be operably linked to p35 through a protease cleavable linker. Alternatively, the sdAb can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody light chain is operably linked to p40 through a protease cleavable linker. The second polypeptide comprises at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the second polypeptide and together with said light chain forms and IL-12 binding site. The protease cleavable linkers in this inducible cytokine prodrug can be the same or different.

[0687]

[0396] In an alternative format, a first polypeptide chain can comprise p35, p40, a sdAb and at least an antigen binding portion of an antibody heavy chain. p35 and p40 can be operably linked, and the sdAb can be operably linked to p40 or through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p35 through a protease cleavable linker. Alternatively, the sdAb can be operably linked to p35 through a protease cleavable linker and the antigen binding portion of an antibody heavy chain can be operably linked to p40 through a second protease cleavable linker. A second polypeptide comprises at least an antigen binding portion of an antibody light chain that is complementary to the heavy chain in the second polypeptide and together with said light chain forms and IL-12 binding site. The protease cleavable linkers in this complex can be the same or different.

[0688]

[0397] Typically, the inducible cytokine prodrugs disclosed herein comprise a blocking domain. The blocking domain can be any element that binds to the cytokine and inhibits the ability of the cytokine polypeptide to bind and activate its receptor. The blocking domain can inhibit the ability of the cytokine (e.g. IL-2, IL-12, or IFN) to bind and / or activate its receptor e.g., by sterically blocking and / or by noncovalently binding to the cytokine polypeptide. The blocking domain disclosed herein can bind to IL-2, IL-12 (e.g. p35, p40, or the heterodimer), or IFN (e.g., IFN-alpha (e.g., human IFN-alpha1, human IFN-alpha2, human IFN-alpha4, human IFN-alpha5, human IFN-alpha6, human IFN-alpha7, human IFN-alpha8, human IFN-alpha10, human IFN-alpha13, human IFN-alpha14, human IFN-alpha16, human IFN-alpha17, human IFN-alpha2) IFN-beta, IFN-gamma).

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[0691]

[0398] Examples of suitable blocking domains include the full length or a cytokine-binding fragment or mutein of the cognate receptor of a cytokine (e.g. IL-2, IL-12, or IFN). The cognate receptor for IL-2 can be the IL-2 alpha chain, the IL-2 beta chain, the IL-2 gamma chain, or combinations thereof. The cognate receptor for IL-12 can be IL-12Rβ1 and / or IL-12Rβ2. The cognate receptor for IFN can be the IFNGR receptor or a portion thereof. For instance, when the interferon polypeptide is an IFNalpha, such as INFalpha2a, the blocking domain can be the extracellular portion of the INFalpha receptor 1 (IFNAR1) or interferon binding portion or mutein thereof, or the extracellular portion of the IFNalpha receptor 2 (IFNAR2) or interferon binding portion or mutein thereof. When the interferon polypeptide is IFNgamma, the blocking domain can be the extracelluar portion of the IFNgamma receptor 1 (IFNGR1) or interferon binding portion or mutein thereof, or the extracellular portion of the IFNgamma receptor 2 (IFNGR2) or interferon binding portion or mutein thereof.

[0692]

[0001] Antibodies and antigen-binding fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like that bind to a cytokine (e.g., IL-2, IL-12, or IFN) can also be used. Other suitable antigen-binding domain that bind to the cytokine polypeptide can also be used, include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds.

[0693]

[0399] Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of the to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides. Blocking domains that are particularly suitable are single chain variable fragments (scFv) or Fab fragments.

[0694]

[0400] The blocking domain can contain two or more components that are present on the same polypeptide chain or on separate polypeptide chains. A first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is

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[0697] complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form an antigen-binding site, such as a Fab that binds the cytokine (e.g., IL-2, IL-12, IFN) and attenuates cytokine activity.

[0698]

[0401] The sdAbs disclosed herein can also act as blocking domains that are capable of blocking all or some of the receptor agonist activity of the cytokine. For instance, the sdAb can contribute to blocking when the sdAb is adjacent to the cytokine polypeptide.

[0699] i. Inducible IL-2 Prodrugs

[0700]

[0402] This disclosure relates to inducible IL-2 prodrugs. The inducible IL-2 prodrugs comprise an IL-2 polypeptide, a sdAb as described herein that extends the half-life of the inducible IL-2 prodrug, an IL-2 blocking domain, and a protease cleavable linker. Preferred inducible IL-2 prodrugs comprise two polypeptide chains.

[0701]

[0403] The first polypeptide chain comprises from amino to carboxy terminus: the IL-2 polypeptide – a protease cleavable linker – a sdAb that binds human serum albumin (HSA) – a linker that is preferably protease cleavable – VH and CH1 of an antibody that binds IL-2. The second polypeptide chain comprises a VL and CL of an antibody that binds IL-2 and that together with the VH and CH1 of the first polypeptide chain form a Fab that binds the IL-2 polypeptide.

[0702]

[0404] For example, the inducible IL-2 prodrug can have a) a first polypeptide of Formulas (I)-(VII), wherein [A] is a IL-2 polypeptide, [D] is an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH), [H] is a sdAb or non-immunoglobulin format that binds HSA as disclosed herein, [L1] is a protease-cleavable polypeptide linker, [L2] is an polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker; and b) a second polypeptide chain comprising an antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH. [L1] and [L2] or [L1] and [L2’] can have the same or different amino acid sequence and or protease-cleavage site (when L2 is protease-cleavable) as desired.

[0703]

[0405] The inducible IL-2 prodrugs comprise a sdAb comprising a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7,

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[0706] CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v)

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[0709] CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0710]

[0406] The inducible IL-2 prodrugs can comprise a sdAb comprising the amino acid sequence of any one of SEQ ID NO: 39-79, 286-290, and an amino acid sequence interest, such an amino acid sequence encoding a cytokine.

[0711]

[0407] The inducible IL-2 prodrug comprises a first polypeptide chain having Formula (I): [A]-[L1]-[H]-[L2]-[D]. In Formula (I), [A] is an IL-2 polypeptide that comprises or consists of SEQ ID NO: 474 or a variant thereof, [L1] is a protease cleavable linker that comprises or consists of SEQ ID NO: 217 or a variant thereof, [H] is a sdAb that binds HSA and comprises or consists of SEQ ID NO: 480 or 39-79, 286-290 or a variant thereof, [L2] is a protease cleavable linker that comprises or consists of SEQ ID NO: 217 or a variant thereof, [D] is an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that comprises or consists of SEQ ID NO: 475 or a variant thereof. The second polypeptide chain comprises an antibody light chain of SEQ ID NO: 476 or a variant thereof that is complementary to [D] (e.g., the VH + CH1) in the first polypeptide and together with the light chain forms an IL-2 binding site.

[0712]

[0408] In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 480 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 39 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 40 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 41 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 42 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 43 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 44 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 45 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 46 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 47 or a variant thereof.

[0713]

[0409] In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 48 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 49 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 50 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 51 or a variant thereof. In Formula (I), the sdAb [H] can comprise 80

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[0715] or consist of SEQ ID NO: 52 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 53 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 54 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 55 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 56 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 57 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 58 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 59 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 60 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 61 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 62 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 63 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 64 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 65 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 66 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 67 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 68 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 69 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 70 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 71 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 72 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 73 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 74 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 75 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 76 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 77 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 78 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 79 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 78 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 286 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 287 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 288 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 289 or a variant thereof. In Formula (I), the sdAb [H] can comprise or consist of SEQ ID NO: 290 or a variant thereof.

[0716]

[0410] In Formula (I), the sdAb [H] can comprise or consists of the amino acid sequence of any of SEQ ID NOs: 39-79 or 286-390, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 39-79 or 286-290. For example, the sdAbs can comprise or consist of an amino acid sequence 81

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[0718] that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 286, 287, 288, 289, or 290.

[0719]

[0411] Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, and 146 are specific examples of inducible IL-2 prodrugs, which are suitable for use according to this disclosure. Compound 117 and additional details regarding its activity are disclosed in International Application No.: WO2021 / 097376 Further details of exemplary inducible IL-2 prodrugs are described in Table 5.

[0720] Table 5. Inducible IL-2 prodrugs

[0721] Inducible IL-2 First Polypeptide Second Polypeptide

[0722] Prodrug

[0723]

[0724] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0725] Compound 16 SEQ ID NO: 341 SEQ ID NO: 343 Compound 17 SEQ ID NO: 342 SEQ ID NO: 343

[0726]

[0727] 83

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[0729] Compound 146 SEQ ID NO: 510 SEQ ID NO: 343

[0730]

[0731] omprises or consists of the amino acid sequence of SEQ ID NO: 326, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0732]

[0413] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 327, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0733]

[0414] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 328, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0734]

[0415] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 329, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0735]

[0416] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 330, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0736]

[0417] Inducible IL-2 prodrug can comprise include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 331, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0737]

[0418] Inducible IL-2 prodrug can comprise can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 332, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0738]

[0419] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 333, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0739]

[0420] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 334, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0740]

[0421] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 335, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0741] 84

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[0743]

[0422] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 336, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0744]

[0423] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 337, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0745]

[0424] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 338, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0746]

[0425] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 339, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0747]

[0426] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 340, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0748]

[0427] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 341, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0749]

[0428] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 342, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0750]

[0429] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 466, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343. Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 482, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0751]

[0430] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 483, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0752]

[0431] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 484, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

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[0755]

[0432] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 485, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0756]

[0433] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 486, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0757]

[0434] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 487, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0758]

[0435] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 488, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0759]

[0436] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 489, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0760]

[0437] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 490, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0761]

[0438] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 491, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0762]

[0439] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 492, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0763]

[0440] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 493, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0764]

[0441] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 494, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

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[0767]

[0442] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 495, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0768]

[0443] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 496, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0769]

[0444] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 497, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0770]

[0445] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 498, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0771]

[0446] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 499, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0772]

[0447] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 500, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0773]

[0448] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 501, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0774]

[0449] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 502, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0775]

[0450] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 503, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0776]

[0451] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 504, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

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[0779]

[0452] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 505, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0780]

[0453] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 506, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0781]

[0454] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 507, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0782]

[0455] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 508, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0783]

[0456] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 509, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0784]

[0457] Inducible IL-2 prodrug can comprise 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 510, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 343.

[0785]

[0458] Amino acid sequence variants of Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 17, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, and 146 that retain attenuated IL-2 activity in the periphery and that release active IL-2 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, the inducible IL-2 prodrugs can include 1) a first polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 326-343, and 2) a second polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical SEQ ID NO: 343.

[0786] ii. Inducible IL-12 Prodrugs

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[0789]

[0459] The inducible cytokine prodrugs can comprise an IL-12 polypeptide. The inducible IL-12 prodrugs comprise an IL-12 polypeptide, a sdAb as described herein that extends the half-life of the inducible IL-2 prodrug, an IL-12 blocking domain, and a protease cleavable linker. Preferred inducible IL-12 prodrugs comprise two polypeptide chains.

[0790]

[0460] In an example, the first polypeptide chain can comprise from amino to carboxy terminus: the p35 IL-12 subunit polypeptide – a protease cleavable linker – a sdAb that binds HSA – a protease cleavable linker – single chain variable fragment (scFv) (i.e., a VH+VL) that binds IL-12. The second polypeptide chain comprises the p40 IL-12 subunit polypeptide. The p35 IL-12 subunit on the first polypeptide chain and the p40 IL-12 subunit on the second polypeptide chain associate to form a biologically active IL-12 heterodimer, which has intrinsic IL-12 receptor agonist activity.

[0791]

[0461] In an example, the IL-12 polypeptide complex comprises a first polypeptide having the formula:

[0792] [A]-[L1]-[H]-[L3]-[D] or [D]-[L3]-[H]-[L1]-[A] or [H]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[H], wherein, [A] is the IL-12 subunit (e.g. p35 or p40); [L1] is the first protease-cleavable linker; [L2] is the second protease cleavable linker; [L3] is the optionally cleavable linker; [H] is a sdAb as disclosed herein and [D] is the blocking domain, and a second polypeptide comprising a IL-12 subunit (e.g., p35 or p40). When the first polypeptide comprises p35 the second polypeptide comprises p40. When the first polypeptide comprises p40 the second polypeptide comprises p35.

[0793]

[0462] In another example, the first polypeptide comprises the formula: [A]-[L1]-[D] or [D]-[L1]-[A]; and the second polypeptide has the formula: [A’]-[L2]-[H] or [H]-[L2]-[A’], wherein [A] is either p35 or p40, wherein when [A] is p35, [A’] is p40 and when [A] is p40, [A’] is p35; [A’] is either p35 or p40; [L1] is the first protease cleavable linker; [L2] is the second protease cleavable linker; [H] is the sdAb; and [D] is the blocking domain.

[0794]

[0463] The inducible cytokine prodrugs can comprise an IL-12 polypeptide. The inducible IL-12 prodrugs comprise an IL-12 polypeptide, a sdAb as described herein that extends the half-life of the inducible IL-12 prodrug, an IL-2 blocking domain, and a protease cleavable linker. Preferred inducible IL-2 prodrugs comprise one polypeptide chain or two polypeptide chains.

[0795]

[0464] The inducible IL-12 prodrugs comprise a sdAb comprising a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or 89

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[0797] consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID 90

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[0799] NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0800]

[0465] The inducible IL-12 prodrug comprises a first polypeptide having the formula: [H]-[L1]-[A]-[L2]-[D].

[0801]

[0466] In this formula, [A] is a p35 IL-2 subunit polypeptide that comprises or consists of SEQ ID NO: 477 or a variant thereof, [L1] is a protease cleavable linker that comprises or consists of SEQ ID NO: 217 or a variant thereof, [H] is a sdAb that binds HSA and comprises or consists of SEQ ID NO: 480, 39-79, or 286-290 or a variant thereof, [L2] is a protease cleavable linker that comprises or consists of SEQ ID NO: 217 or a variant thereof, [D] is scFv (VH + VL) that binds IL-12 that comprises or consists of SEQ ID NO: 478 or a variant thereof. The second polypeptide chain comprises a p40 IL-12 subunit polypeptide of SEQ ID NO: 444. The p35 IL-12 subunit on the first polypeptide chain and the p40 IL-12 subunit on the second polypeptide chain associate to form a biologically active IL-12 heterodimer, which has intrinsic IL-12 receptor agonist activity.

[0802]

[0467] In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 48 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 49 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 50 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 51 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 52 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 53 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 54 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 55 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 56 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 57 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 58 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 59 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 60 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 61 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 62 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 63 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 64 or a 91

[0803] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0804] variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 65 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 66 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 67 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 68 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 69 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 70 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 71 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 72 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 73 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 74 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 75 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 76 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 77 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 78 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 79 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 329 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 330 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 332 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 79 or a variant thereof. In the formula, the sdAb [H] can comprise or consist of SEQ ID NO: 333 or a variant thereof.

[0805]

[0468] In the, the sdAb [H] can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 39-79 or 286-390, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 39-79 or 286-390. For example, the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 286, 287, 288, 289, 290.

[0806]

[0469] The inducible IL-12 prodrugs can comprise a sdAb comprising the amino acid sequence of any one of SEQ ID NO: 39-79 or 426-430, and an amino acid sequence interest, such an amino acid sequence encoding a cytokine.

[0807] 92

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[0809]

[0470] Compounds 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67 are specific examples of inducible IL-12 prodrugs that comprise a single polypeptide chain.

[0810] Compounds 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 147-193 are specific examples of inducible IL-12 prodrugs that comprise two polypeptide chains. Compound 127 and additional details regarding its activity are disclosed in International Application No.: PCT / US2021 / 33014 (WO2021 / 236676). Further details of exemplary inducible IL-12 prodrugs are described in Table 6.

[0811] Table 6. Inducible IL-12 Prodrugs

[0812] Inducible IL-12 Prodrugs First Polypeptide Second Polypeptide

[0813] Compound 18 SEQ ID NO: 344 N / A

[0814]

[0815] \\4151-9465-8144 v1 Attorney Docket No.: 761146.252320

[0816] Compound 38 SEQ ID NO: 364 N / A Compound 39 SEQ ID NO: 365 N / A

[0817]

[0818] 94

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[0820] Compound 69 SEQ ID NO: 395 SEQ ID NO: 444 Compound 70 SEQ ID NO: 396 SEQ ID NO: 444

[0821]

[0822] 95

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[0824] Compound 99 SEQ ID NO: 426 SEQ ID NO: 444 Compound 100 SEQ ID NO: 427 SEQ ID NO: 444

[0825]

[0826] 96

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[0828] Compound 158 SEQ ID NO: 522 SEQ ID NO: 444 Compound 159 SEQ ID NO: 523 SEQ ID NO: 444

[0829]

[0830] 97

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[0832] Compound 184 SEQ ID NO: 548 SEQ ID NO: 444

[0833] Compound 185 SEQ ID NO: 549 SEQ ID NO: 444

[0834]

[0835] sequence of SEQ ID NO: 344. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 345. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 346. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 347. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 348. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 349. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 350. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 351. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 352. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 353. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 354. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 355. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 356. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 357. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 358. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 359. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 360. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 361. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO:

[0836] 98

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[0838] 362. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 363. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 364. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 365. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 366. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 367. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 368. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 369. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 370. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 371. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 372. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 373. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 374. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 375. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 376. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 377. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 378. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 379. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 380. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 381. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 382. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 383. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 384. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 385. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 386. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 387. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 388. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 389. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 390. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 391. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 392. The inducible IL-12 prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 393.

[0839] 99

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[0841]

[0472] Amino acid sequence variants of Compounds 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67 that retain attenuated IL-12 activity in the periphery and that release active IL-12 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure.

[0842]

[0473] As disclosed herein, the inducible IL-12 prodrugs can comprise two polypeptide chains.

[0843]

[0474] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 394, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0844]

[0475] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 395, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0845]

[0476] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 396, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0846]

[0477] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 397, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SE Q ID NO: 444.

[0847]

[0478] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 398, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0848]

[0479] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 399, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0849]

[0480] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 400, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0850]

[0481] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 401, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0851] 100

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[0853]

[0482] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 402, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0854]

[0483] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 403, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0855]

[0484] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 404, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0856]

[0485] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 405, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0857]

[0486] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 406, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0858]

[0487] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 407, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0859]

[0488] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 408, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 268.

[0860]

[0489] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 409, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0861]

[0490] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 410, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0862]

[0491] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 411, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0863] 101

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[0865]

[0492] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 412, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0866]

[0493] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 413, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0867]

[0494] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 414, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0868]

[0495] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 415, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0869]

[0496] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 416, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0870]

[0497] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 417, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0871]

[0498] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 418, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0872]

[0499] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 419, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0873]

[0500] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 420, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0874]

[0501] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 421, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0875] 102

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[0877]

[0502] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 422, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0878]

[0503] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 423, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0879]

[0504] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 424, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0880]

[0505] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 425, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0881]

[0506] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 426, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0882]

[0507] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 427, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0883]

[0508] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 428, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0884]

[0509] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 429, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0885]

[0510] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 430, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0886]

[0511] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 431, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0887] 103

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[0889]

[0512] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 432, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0890]

[0513] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 433, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0891]

[0514] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 434, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0892]

[0515] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 435, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0893]

[0516] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 436, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0894]

[0517] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 437, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0895]

[0518] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 438, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0896]

[0519] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 439, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0897]

[0520] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 440, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0898]

[0521] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 441, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0899] 104

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[0901]

[0522] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 442, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0902]

[0523] The inducible IL-12 prodrug can include 1) a first polypeptide that comprises or consists of the amino acid sequence of SEQ IN NO: 443, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0903]

[0524] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 511, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0904]

[0525] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 512, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0905]

[0526] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 513, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0906]

[0527] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 514, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0907]

[0528] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 515, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0908]

[0529] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 516, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0909]

[0530] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 517, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0910]

[0531] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 518, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0911] 105

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[0913]

[0532] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 519, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0914]

[0533] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 520, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0915]

[0534] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 521, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0916]

[0535] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 522, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0917]

[0536] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 523, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0918]

[0537] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 524, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0919]

[0538] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 525, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0920]

[0539] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 526, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0921]

[0540] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 527, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0922]

[0541] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 528, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

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[0925]

[0542] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 529, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0926]

[0543] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 530, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0927]

[0544] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 531, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0928]

[0545] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 532, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0929]

[0546] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 533, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0930]

[0547] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 534, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0931]

[0548] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 535, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0932]

[0549] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 536, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0933]

[0550] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 537, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0934]

[0551] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 538, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

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[0937]

[0552] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 539, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0938]

[0553] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 540, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0939]

[0554] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 541, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0940]

[0555] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 542, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0941]

[0556] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 543, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0942]

[0557] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 544, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0943]

[0558] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 545, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0944]

[0559] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 546, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0945]

[0560] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 547, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0946]

[0561] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 548, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

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[0949]

[0562] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 549, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0950]

[0563] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of amino acid sequence of SEQ ID NO: 550, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0951]

[0564] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 551, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0952]

[0565] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 552, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0953]

[0566] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 556, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0954]

[0567] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 557, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0955]

[0568] The inducible IL-12 prodrug can comprise 1) a first polypeptide that comprises or consists of an amino acid sequence of SEQ ID NO: 560, and 2) a second polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 444.

[0956]

[0569] Amino acid sequence variants of Compounds 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, or 147-193 that retain attenuated IL-12 activity in the periphery and that release active IL-12 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, the inducible IL-12 prodrugs can include 1) a first polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 394-443 511-552, 556, 557, or 560, and 2) a second polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least

[0957] 109

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[0959] about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical SEQ ID NO: 444.

[0960] iii. Inducible IFN Prodrugs

[0961]

[0570] The inducible cytokine prodrugs can comprise an IFN. The inducible IFN prodrugs comprise an IFN polypeptide, a sdAb as described herein that extends the half-life of the inducible IFN prodrug, a protease cleavable linker, and optionally a blocking domain. Where the blocking domain is absent, it is preferred that the sdAb also functions as a blocking domain, e.g., the sdAb sterically inhibits binding of the IFN polypeptide in the prodrug to the IFN receptor. When the blocking domain is absent, two or more sdAbs can also function as a blocking domain to sterically inhibit binding of the IFN polypeptide in the prodrug to the IFN receptor. The inducible IFN polypeptide can be operably linked to the sdAb, the blocking domain (when present), or both the sdAb and the blocking domain (when present) by a protease cleavable linker. Typically, the single polypeptide chain comprises one or two IFN polypeptides. The IFN polypeptide can be located at any desired position.

[0962]

[0571] The inducible IFN prodrugs comprise a sdAb comprising a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ 110

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[0964] ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0965]

[0572] The inducible IFN prodrugs can comprise a sdAb comprising the amino acid sequence of any one of SEQ ID NO: 39-79 or 426-430, and an amino acid sequence interest, such an amino acid sequence encoding a cytokine.

[0966]

[0573] Compounds 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, and 132, are specific examples of inducible IFN prodrugs. Further details of exemplary inducible IFN prodrugs is described in Table 7.

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[0969] Table 7. Inducible IFN Prodrugs

[0970] Inducible IFN Prodrugs First Polypeptide Second Polypeptide Cd 117 SE ID NO 446 NA

[0971]

[0972]

[0001] The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 446. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 447. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 448. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 449. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 450. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 451. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 452. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 453. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 454. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 455. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 456. The 112

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[0974] inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 457. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 458. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 459. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 460. The inducible IFN prodrug can comprise or consist of the amino acid sequence of SEQ ID NO: 461.

[0975]

[0002] Amino acid sequence variants of Compounds 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, and 132 that retain attenuated IFN activity in the periphery and that release active IFN upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, the inducible IL-12 prodrugs can comprise or consist of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 446-461.

[0976] G. Pharmaceutical Compositions

[0977]

[0574] The disclosure further relates to pharmaceutical compositions comprising a sdAb and non-immunoglobulin formats disclosed herein, fusion polypeptides or conjugates as disclosed herein, or inducible cytokine prodrugs as disclosed herein. The pharmaceutical formulations or compositions comprise a sdAbs described herein, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein and typically a pharmaceutically acceptable carrier.

[0978]

[0575] Compositions comprising the sdAbs described herein, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein are suitable for administration in vitro or in vivo. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the subject to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0979]

[0576] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an 113

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[0981] appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides.

[0982] Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are suitable for administration of the sdAbs described herein, the fusion polypeptides or conjugates described herein, inducible cytokine prodrugs disclosed herein or nucleic acid sequences encoding the sdAbs, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein to humans or other subjects.

[0983]

[0577] In some embodiments of the pharmaceutical compositions, the sdAbs described herein, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the sdAbs, the fusion polypeptides or conjugates as described herein, or inducible cytokine prodrugs disclosed herein. In some instances, the sdAbs, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein are conjugated to the surface of liposomes. In some instances, the sdAbs as described herein, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0984]

[0578] The sdAbs described herein, the fusion polypeptides or conjugates described herein, or inducible cytokine prodrugs disclosed herein are suitable for use as a medicament and can be administered by an appropriate route of administration, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

[0985] 114

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[0987]

[0579] While parenteral administration is generally preferred, optionally, the sdAbs described herein, the fusion polypeptides or conjugates described herein, the inducible cytokine prodrugs disclosed herein or nucleic acid sequences encoding the sdAbs, the fusion polypeptides or conjugates, or inducible cytokine prodrugs disclosed herein are administered by a vector. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo via, for example, expression v...

Claims

1. Attorney Docket No.: 761146.2523202.CLAIMS1. A polypeptide comprising:4.a) a sdAb that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3; wherein5.CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions;6.CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and7.CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and8.b) an amino acid encoding a polypeptide with biological activity; wherein in the polypeptide with biological activity is a tumor antigen binding domain, a cytokine, with the proviso that the cytokine is not IL-21, a chemokine, a growth factor, a soluble receptor, a peptide hormone, a hormone receptor agonist, or muteins, variants or combinations of any of the foregoing.

2. The polypeptide of claim 1, wherein the tumor antigen binding domain binds EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA.5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, IR WT1.1.

3. The polypeptide of claim 1, wherein the cytokine is IL-2, IL-12, IL-10, IL-15, IL-18, IL-23, IL-36, IFN, IL-7, Tumor Necrosis Factor (TNF), Granulocyte macrophage colony-stimulating factor (GM-CSF), or a functional fragment or mutein of any of the foregoing.

4. The polypeptide of claim 1, wherein the chemokine is CXCL10, CCL19, CCL20, CCL21, or functional fragment of any of the foregoing.12.32413.\\4151-9465-8144 v1 Attorney Docket No.: 761146.2523205. The polypeptide of any one of the preceding claims, further comprising an optionally cleavable linker, wherein the optionally cleavable linker is operably linked to the sdAb and the amino acid sequence of interest.

6. The polypeptide of claim 5, wherein the optionally cleavable linker is cleavable.

7. The polypeptide of claim 5, wherein the optionally cleavable linker comprises a cleavable moiety that is a substrate for one or more proteases.

8. The polypeptide of claim 5, wherein the optionally cleavable linker is not cleavable.

9. The polypeptide of any one of claims 5-8, wherein the protease cleavable linker comprises a sequence that is capable of being cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a caspase, a tryptase, or a tumor protease.

10. The polypeptide of any one of claims 1 or 2, wherein the polypeptide further comprises an immune cell binding domain.

11. The polypeptide of claim 10, wherein the immune cell binding domain has specificity for an antigen on a T-cell.

12. The polypeptide of claim 11, wherein the antigen on the T-cell is CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta.

13. The polypeptide of any one of claims 1, 2, or 10-12, wherein the polypeptide with biological activity is a bispecific engager, wherein the bispecific engager comprises a first binding region with specificity on a T cell and a second binding region with specificity for a tumor associated antigen.

14. The polypeptide of any one of the preceding claims, wherein the protease cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214-239 or an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 214-239.24.32525.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232015. The polypeptide of any one the preceding claims, wherein the protease cleavable linker comprises an amino acid sequence of SEQ ID NO: 214 or SEQ ID NO: 217.

16. The polypeptide any one of the preceding claims, wherein the sdAb comprises:28.(a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 amino acid substitutions;29.(b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions;30.(c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;31.(d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;32.(e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;33.(f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR334.32635.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232036.comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;37.(g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;38.(h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;39.(i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;40.(j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;41.(k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;42.(l) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;43.32744.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232045.(m) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; or46.(n) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

17. The polypeptide of any one of the preceding claims, wherein the sdAb comprises:48.(a) a CDR1 that consists of SEQ ID NO: 7; a CDR2 that consists of SEQ ID NO: 38; and a CDR3 that consists of SEQ ID NO: 12;49.(b) a CDR1 that consists of SEQ ID NO: 37; a CDR2 that consists of SEQ ID NO: 9; and a CDR3 that consists of SEQ ID NO: 12;50.(c) a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 10; and a CDR3 that consists of SEQ ID NO: 12;51.(d) a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 11; and a CDR3 that consists of SEQ ID NO: 12;52.(e) a CDR1 that consists of SEQ ID NO: 1; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 3; or53.(f) a CDR1 that consists of SEQ ID NO: 4; a CDR2 that consists of SEQ ID NO: 5; and a CDR3 that consists of SEQ ID NO: 6.

18. The polypeptide of any one of the preceding claims, wherein the sdAb is a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain.

19. The polypeptide of any one of the preceding claims, wherein the sdAb is a variable heavy domain of heavy chain (VHH).56.32857.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232020. The polypeptide of any one of any one of the preceding claims, wherein the sdAb comprises the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290.

21. The polypeptide of any one of the preceding claims, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 39.

22. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 40.

23. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 42.

24. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 49.

25. The polypeptide of any one of claims 1-20, wherein the s sdAb comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 53.

26. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 55.

27. The polypeptide of any one of claims 1-20, wherein the v comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 56.66.32967.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232028. The polypeptide of any one of claims 1-20, wherein the comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 57.

29. The polypeptide of any one of claims 1-20, wherein the sdAb does not comprises the amino acid sequence of SEQ ID NO: 59.

30. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 63.

31. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 66.

32. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 68.

33. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 73.

34. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 75.

35. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 76.76.33077.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232036. The polypeptide of any one of claims 1-20, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 77.

37. A nucleic acid encoding the polypeptide of any one of the preceding claims.

38. The nucleic acid of claim 37, wherein the nucleic acid comprises a circular vector.

39. The nucleic acid of any one of claims 37 or 39, wherein the nucleic acid comprises DNA or RNA.

40. A vector comprising the nucleic acid of any one of claims 37-39.

41. A host cell comprising the vector of claim 40.

42. A method of making a pharmaceutical composition, comprising culturing the host cell of claim 36 under suitable conditions for expression of the polypeptide.

43. The method of claim 42, further comprising isolating the polypeptide.

44. A pharmaceutical composition comprising a polypeptide of any one of claims 1-36, or nucleic acid of any one of claims 37-39.

45. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the polypeptide of any one of claims 1-36.

46. An inducible cytokine prodrug comprising at least one of each of:89.a) a cytokine polypeptide [A] with the proviso that the cytokine polypeptide is not IL-21; b) a single domain antibody fragment (sdAb) [H] that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3; wherein90.CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and CDR391.33192.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232093.comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions;94.c) a protease cleavable linker [L]; and95.d) optionally a cytokine blocking domain that binds the cytokine polypeptide and inhibits the receptor activating activity of the cytokine polypeptide [D].

47. The inducible cytokine prodrug of claim 46, wherein the optional cytokine blocking domain comprises a ligand-binding domain or fragment of a cognate receptor for the cytokine polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the cytokine polypeptide.

48. The inducible cytokine prodrug of claim 47, wherein the antibody or antigen-binding fragment is a single domain antibody, a Fab, or a scFv that binds the cytokine polypeptide.

49. The inducible cytokine prodrug of any one of claims 46-48, wherein the cytokine polypeptide is IL-2, IL-10, IL-12, IL-15, IL-18, IL-10, IL23, IL-36, IFNalpah, IFNbeta, IFNgamma, IL-7, Tumor Necrosis Factor (TNF), Granulocyte macrophage colony-stimulating factor (GM-CSF), or a functional fragment or mutein of any of the foregoing.

50. The inducible cytokine prodrug of claim 49, wherein the cytokine polypeptide is IL-2.

51. The inducible cytokine prodrug of claim 49, wherein the cytokine polypeptide is IL-12.

52. The inducible cytokine prodrug of any one of claims 46-51, wherein the protease cleavable linker comprises a sequence that is capable of being cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a caspase, a tryptase, or a tumor protease.

53. The inducible cytokine prodrug of any one of claims 46-52, wherein the protease cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214-239 or an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 214-239.103.332104.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232054. The inducible cytokine prodrug of any one claims 46-52, wherein the protease cleavable linker comprises an amino acid sequence of SEQ ID NO: 217 or SEQ ID NO: 214.

55. The inducible cytokine prodrug of any one claims 46-52, wherein the sdAb comprises:107.(a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 amino acid substitutions;108.(b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions;109.(c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;110.(d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;111.(e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;112.(f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3113.333114.\\4151-9465-8144 v1 Attorney Docket No.: 761146.252320115.comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;116.(g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;117.(h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;118.(i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;119.(j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;120.(k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;121.(l) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;122.334123.\\4151-9465-8144 v1 Attorney Docket No.: 761146.252320124.(m) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; or125.(n) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

56. The inducible cytokine prodrug of any one claims 46-55, wherein the sdAb comprises:127.(a) a CDR1 that consists of SEQ ID NO: 7; a CDR2 that consists of SEQ ID NO: 38; and a CDR3 that consists of SEQ ID NO: 12;128.(b) a CDR1 that consists of SEQ ID NO: 37; a CDR2 that consists of SEQ ID NO: 9; and a CDR3 that consists of SEQ ID NO: 12;129.(c) a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 10; and a CDR3 that consists of SEQ ID NO: 12;130.(d) a CDR1 that consists of SEQ ID NO: 8; a CDR2 that consists of SEQ ID NO: 11; and a CDR3 that consists of SEQ ID NO: 12;131.(e) a CDR1 that consists of SEQ ID NO: 1; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 3; or132.(f) a CDR1 that consists of SEQ ID NO: 4; a CDR2 that consists of SEQ ID NO: 5; and a CDR3 that consists of SEQ ID NO: 6.

57. The inducible cytokine prodrug of any one of the claims 46-56, wherein the sdAb is a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain.

58. The inducible cytokine prodrug of any one of the claims 46-57, wherein the sdAb is a variable heavy domain of heavy chain (VHH).135.335136.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232059. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 39-58, 60-79, or 286-290.

60. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 39.

61. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 40.

62. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 42.

63. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 49.

64. The inducible cytokine prodrug of any one of the claims 46-58, wherein the s sdAb comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 53.

65. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 55.

66. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 56.145.336146.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232067. The inducible cytokine prodrug of any one of the claims 46-58, wherein the comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 57.

68. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb does not comprises the amino acid sequence of SEQ ID NO: 59.

69. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 63.

70. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 66.

71. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 68.

72. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 73.

73. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 75.

74. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 76.155.337156.\\4151-9465-8144 v1 Attorney Docket No.: 761146.25232075. The inducible cytokine prodrug of any one of the claims 46-58, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 77.

76. A nucleic acid encoding the inducible cytokine prodrug of any one of claims 46-75.

77. A vector comprising the nucleic acid of claim 76.

78. A host cell comprising the vector of claim 77.

79. A method of making a inducible cytokine prodrug of any one of claims 46-75, comprising culturing the host cell of claim 73 under suitable conditions for expression of the inducible cytokine prodrug.

80. The method of claim 79, further comprising isolating the inducible cytokine prodrug.

81. A pharmaceutical composition comprising a inducible cytokine prodrug of any one of claims 46-75, or nucleic acid of claim 77.

82. A pharmaceutical composition comprising an inducible cytokine prodrug of any one of claims 46-75, or nucleic acid of claim 77.

83. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the inducible cytokine prodrug of any one of claims 46-75.166.338167.\\4151-9465-8144 v1

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